Compositions and methods for bacterial production of chondroitin

ABSTRACT

The invention relates to the field of recombinant DNA technology for the production of chondroitin, including the production of chondroitin sulfate via a combination of recombinant bacterial fermentation and post-fermentation sulfation.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S. Appl. No.61/309,407, filed Mar. 1, 2010, which is hereby incorporated byreference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The content of the electronically submitted sequence listing(“sequencelisting_ascii.txt”, 902,707 bytes, created on Mar. 1, 2011)filed with the application is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of recombinant DNA technology for theproduction of chondroitin, including the production of chondroitinsulfate via a combination of recombinant bacterial fermentation andpost-fermentation sulfation.

2. Background Art

Chondroitin belongs to a family of heteropolysaccharides calledglycosaminoglycans (GAGs). Glycosaminoglycans (GAGs) are unbranched,negatively charged polysaccharide chains composed of repeatingdisaccharide units, one of which is an acidic sugar and the other ofwhich is an amino sugar (N-acetylglucosamine or N-acetylgalactosamine),that can be sulfated. Because of their inflexible nature and highnegative charge, GAGs exhibit highly extended conformations occupyinglarge amounts of space, attracting cations and water and forming porousgels in the extracellular matrix. As such, GAGs, which are found in mostanimals, help to hydrate and expand tissues and enable the matrix towithstand compressive forces. The cartilage matrix lining the kneejoint, for example, can support pressures of hundreds of atmospheres bythis mechanism.

Chondroitin sulfate is important for maintenance of cartilage strengthand resilience and is marketed as a nutritional supplement to reducejoint pain and promote healthy cartilage and joint function. Clinicalstudies support the use of chondroitin sulfate for treatment ofosteoarthritis (See, for example, Kahan et al., Arthritis and Rheumatism2009; 60:524-533; Michel et al., Arthritis and Rheumatism 2005;52:779-786 and Uebelhardt et al., Osteoarthritis and Cartilage 2004;12:269-276), interstitial cystitis (See, for example, Nickel et. al.,BJU Int. 2009; 103:56-60 and Cervigni et al., Int. Urogvnecol. J. PelvicFloor Dysfunct. 2008; 19:943-947), and synovitis (See, for example,Hochberg and Clegg, Osteoarthritis and Cartilage 2008; 16(Suppl.3):S22-S24 and Möller, Osteoarthritis and Cartilage 2009; 17(Suppl.1):S32-S33). These documents are incorporated by reference herein inthere entireties.

Chondroitin sulfate is currently produced by extraction from cartilageof animals including cows, pigs, sharks and poultry, using chemical andenzymatic treatments to dissociate the polysaccharide from the proteinand produce a polysaccharide product of varying quality (Barnhill etal., J. Am. Pharm. Assoc. 2006; 46:14-24, Volpi, J. Pharm. Pharmacol.2009; 61:1271-1280).

Chondroitin contains D-glucuronic acid (GlcUA) andN-acetyl-D-galactosamine (GalNAc). It is composed of a disacchariderepeating unit β3GalNAc-β4GlcUA. Typically, the GalNAc residues arevariably sulfated at the 4 and 6 positions. Chondroitin sulfate occursnaturally as a component of proteoglycans that are structural componentsof cartilaginous tissue, such as joints, in humans and other animals.Proteoglycans consist of a core protein and a polysaccharide component,such as chondroitin sulfate, which is covalently attached to the proteinthrough an oligosaccharide linker as shown in FIG. 1. The core proteinis decorated with multiple polysaccharide chains. Proteoglycans can beanchored in the cell membrane with the polysaccharide-containing portionof the protein present in the extracellular space or can be secreted andlocalized in the extracellular matrix (Prydz and Dalen, J. Cell Sci.2000; 113:193-205).

The glycosyltransferase enzymes responsible for synthesizing thechondroitin backbone (chondroitin synthases) do so by adding alternatingmonosaccharide units of GalNAc and GlcUA from UDP-GalNAc and UDP-GlcUAdonors to an accepting substrate. Theses enzymes have been identified inhumans (Kitagawa et al., J. Biol. Chem. 2001; 276:43894-43900; Yada etal., J. Biol. Chem. 2003; 278:39711-39725), and homologs of humanchondroitin synthase have been identified in a variety of other animalsincluding horse, cow, rodents, dog, chicken, zebra fish, nematodes, andinsects (www.ncbi.nlm.nih.gov/homologene/8950).

Some bacteria also produce chondroitin or chondroitin-likepolysaccharide polymers as a component of their capsule. Unlike thechondroitin sulfate found in vertebrates, microbial chondroitin is notpresent as a proteoglycan, but rather as a lipid-linked polysaccharideon the bacterial cell surface and as free (i.e., not cell-associated)polysaccharide in culture media (Whitfield, Annu. Rev. Biochem. 2006;75:39-68; DeAngelis, Glcobiol. 2002; 12:9R-16R).

Two bacteria, E. coli K4 (Rodriguez et al., Eur. J. Biochem. 1988;177:117-124) and Pasteurella multocida serotype F (Rimler, Vet. Rec.1994; 134:191-192), were reported to produce non-sulfated,chondroitin-like, capsular polysaccharides that potentially could bechemically modified to produce chondroitin sulfate. E. coli K4 was shownby Rodriguez et al. to produce an unsulfated chondroitin backbone withfructose side branches (K4 antigen) as a capsular polymer component.Ninomiya et al. (J. Biol. Chem. 2002; 277:21567-21575) identified andsequenced key genes required for biosynthesis of the chondroitin-likecapsular polysaccharide in E. coli K4. These sequences were depositedwith GenBank™ having accession number AB079602. The sequences disclosedby Ninomiya et al. comprise the so-called “region 2” portion of the“group 2” capsule gene cluster of E. coli K4. A detailed description ofthe organization of capsule gene clusters in E. coli is provided byWhitfield (Annu. Rev. Biochem. 2006; 75:39-68). The region 2 genes of E.coli group 2 capsule gene clusters encode the proteins that determinethe structure of the capsular polysaccharide. The AB079602 sequenceincludes a gene, termed kfoC, that encodes the E. coli K4 chondroitinpolymerase. The E. coli K4 chondroitin polymerase is a bifunctionalglycosyltransferase that transfers GlcUA and GalNAc alternately to thenon-reducing end of a chondroitin saccharide chain and relatedoligosaccharides, producing the chondroitin backbone of the K4 antigenpolysaccharide. The chondroitin-like capsule polysaccharide produced byE. coli K4 contains fructose, linked (β1,3) to the GlcUA residues ofchondroitin. Pasteurella multocida Type F also produces an unsulfatedchondroitin capsule component and the glycosyltransferase responsiblefor chondroitin polymerization in this organism has also been cloned, asreported in DeAngelis & Padgett-McCue, J. Biol. Chem. 2000;275:24124-29. Like the K4 chondroitin polymerase, the Pasteurellachondroitin synthase (pmCS, Genbank Accession No. AAF97500) is a singlepolypeptide enzyme that can synthesize a chondroitin polymer fromUDP-GlcUA and UDP-GalNAc when provided with an appropriate acceptorsubstrate.

Traditional methods of chondroitin sulfate production involvingpurification from animal tissue can be laborious and cost intensive.Moreover, production of chondroitin sulfate from animal tissue isnecessarily associated with the potential for infectious agents to bepresent in the chondroitin sulfate products. Care must be taken duringproduction from animal tissues to minimize the likelihood of suchpotential infectious agents. Such shortcomings can be addressed by usingalternative approaches utilizing recombinant DNA technology forproduction of chondroitin. Recently, microbial production of chondroitinhas been suggested by DeAngelis (US Patent Application Publication No.20030109693) and by Cimini et al. (Appl. Microbiol. Biotechnol. 2010;85(6):1779-87 (Epub Oct. 1, 2009)). However, the known microorganismsthat produce chondroitin (Pasteurella multocida) or chondroitin-like (E.coli K4) polysaccharides are known pathogens to various mammals andtherefore unsuitable for large scale fermentation. They are alsorelatively low producers of the polysaccharide.

In particular, P. multocida is considered not suitable for commercialproduction of chondroitin because of its low yield, requirement ofexpensive media, and Biohazard Level 2 (BL2) status, which requiresspecialized and expensive facilities. High yields from a microorganismwould be necessary for commercially profitable production ofchondroitin. DeAngelis (US Patent Application Publication No.20030109693) mentions the possibility of expressing pmCS in host cellssuch as a food grade Lactococcus or Bacillus to synthesize recombinantchondroitin. However, Bacillus is a gram positive bacterium and, assuch, has a very different membrane/cell wall structure than gramnegative organisms like E. coli and Pasteurella multocida. Efficientsecretion of the polymer would, therefore, be expected to be problematicin Bacillus.

E. coli K4 is also unsuitable for production of chondroitin because itis known to be a human pathogen. Moreover, it does not producechondroitin per se, but instead produces, as noted above, afructosylated form of chondroitin. Extensive chemical or enzymaticmodification of this polysaccharide is required to produce chondroitin.Such modification increases the total cost of the process. Additionally,it requires introduction of further processes and quality controlmeasures to determine that such modification was complete and generateda consistent product.

There is a need, therefore, for an efficient, safe and cost effectiveprocess for the production of chondroitin. The present inventionaddresses this need by providing constructs and host cells and methodsfor recombinant microbial production of chondroitin which cansubsequently be sulfated to produce chondroitin sulfate.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a construct comprising a genecluster comprising kpsF, kpsE, kpsD, kpsU, kpsC, kpsS, kfoA, kfoC, andkfoF, wherein the gene cluster does not contain a functional gene of oneor more of kfoD, orf3(kfoI), kfoE, or orf1(kfoH), and wherein theconstruct is suitable for producing a chondroitin in a non-pathogenicbacterial host cell. In some embodiments, the chondroitin isnon-fructosylated. In some embodiments, the chondroitin is secreted fromthe host cell. In some embodiments, the gene cluster further compriseskfoG, kfoB, or both kfoG and kfoB. In some embodiments, the gene clusterfurther comprises kfoM and kfoT. In some embodiments, the constructcomprises pDD66, pDD67, pCX040, pCX041, pCX042, pCX043, pCX096, orpBR1052.

The present invention is directed to a construct comprising a genecluster comprising kfoA, kfoC, and kfoF, wherein the gene cluster doesnot contain a functional gene of one or more of kpsM, kpsT, kpsE, kpsD,kpsC, or kpsS, and wherein the construct is suitable for producing achondroitin in a non-pathogenic bacterial host cell. In someembodiments, the chondroitin is not secreted from the host cell. In someembodiments, the construct also does not contain a functional gene ofone or more of kfoD, orf3, kfoE, or orf1. In some embodiments, thechondroitin is non-fructosylated. In some embodiments, the gene clusterfurther comprises kfoG, kfoB, or both kfoG and kfoB. In someembodiments, the construct comprises kfoA, kfoB, kfoC, kfoF, and kfoG.In some embodiments, the construct comprises pCX039, pCX044, or pCX092.In some embodiments, the construct comprises pCX045 or pCX048.

The present invention is directed to a construct comprising a geneselected from the group consisitng of kfoA, kfoB, kfoC, kfoF, kfoG, anda combination thereof, wherein the construct does not contain afunctional gene of one or more of kpsM, kpsT, kpsE, kpsD, kpsC, or kpsS,and wherein the construct is suitable for producing a chondroitin orincreasing the amount of a chondroitin in a non-pathogenic bacterialhost cell. In some embodiments, the chondroitin is not secreted from thehost cell. In some embodiments, the construct also does not contain afunctional gene of one or more of kfoD, orf3, kfoE, or orf1. In someembodiments, the chondroitin is non-fructosylated. In some embodiments,the construct comprises kfoA, kfoB, kfoC, kfoF, and kfoG. In someembodiments, the construct comprises pCX075, pCX081, pCX082, pCX092,pCX101, pBR1102, pBR1100 or pBR1101. In some embodiments, the constructcomprises pCX045 or pCX048.

In some embodiments, one or more genes in any of the constructs of theinvention are modified for optimal codon usage in the bacterial hostcell.

In some embodiments, any of the constructs of the invention furthercomprise a promoter. In some embodiments, the promoter is selected fromthe group consisting of Pm, Plac, Ptrp, Ptac, λpL, PT7, PphoA, ParaC,PxapA, Pcad, and PrecA. In some embodiments, the promoter is Pm.

In some embodiments, any of the constructs of the invention furthercomprise a second promoter. In some embodiments, the second promoter isselected from the group consisting of Pm, Plac, Ptrp, Ptac, λpL, PT7,PphoA, ParaC, PxapA, Pcad, and PrecA. In some embodiments, the secondpromoter is Pm. In some embodiments, the second promoter is operablylinked to one or more genes in the construct. In some embodiments, thesecond promoter is operably linked to kpsFEDUCS.

In some embodiments, the construct further comprises a xylS regulatorygene.

In some embodiments, any of the constructs of the invention furthercomprise an antibiotic resistance gene. In some embodiments, theantibiotic resistance gene is selected from the group consisting ofchloramphenicol (CamR), kanamycin (KanR), ampicillin (AmpR),tetracycline (TetR), bleomycin (BleR), spectinomycin (SpcR), sulfonamide(SuR), streptomycin (StrR), carbenicillin (CbR), and erythromycin(EryR).

In some embodiments, any of the constructs of the invention comprise aK4 gene cluster.

In some embodiments, any of the constructs of the invention are suitablefor producing a chondroitin in a non-pathogenic bacterial host cell,wherein the bacterial host cell is or is derived from a non-pathogenicorganism selected from the group consisting of Escherichia, Pseudomonas,Xanthomonas, Methylomonas, Acinetobacter, and Sphingomonas.

The present invention is directed to a non-pathogenic bacterial hostcell comprising any of the constructs of the invention. In someembodiments, the bacterial host cell is or is derived from anon-pathogenic organism selected from the group consisting ofEscherichia, Pseudomonas, Xanthomonas, Methylomonas, Acinetobacter, andSphingomonas. In some embodiments, the bacterial host cell is abacterial strain selected from the group consisting of MSC279, MSC280,MSC315, MSC316, MSC317, MSC319, MSC322, MSC323, MSC324, MSC325, MSC326,MSC328, MSC346, MSC347, MSC348, MSC350, MSC356, MSC359, MSC392, MSC402,MSC403, MSC404, MSC405, MSC410, MSC411, MSC436, MSC437, MSC438, MSC439,MSC458, MSC459, MSC460. MSC461, MSC466 MSC467. MSC469, MSC480, MSC494,MSC498, MSC499, MSC500, MSC510, MSC511, MSC522, MSC526, MSC537, MSC551,MSC561, MSC562, MSC563, MSC564, MSC566, MSC567, MSC619, MSC625, MSC627,MSC640, MSC641, MSC643, MSC646. MSC650, MSC656. MSC657, MSC658, MSC659,MSC660, MSC669, MSC670, MSC671, MSC672, MSC673 MSC674, MSC675, MSC676,MSC677, MSC678, MSC679, MSC680, MSC681, MSC682, MSC683, MSC684, MSC687,MSC688, MSC689, MSC690, MSC691, MSC692, MSC693, MSC694, MSC700, MSC701,MSC702, MSC722, MSC723 and MSC724.

The present invention is directed to a method for producing achondroitin, comprising transferring any of the constructs of theinvention to a non-pathogenic bacterial host cell, and culturing thebacterial host cell under fermentation conditions wherein thechondroitin is produced by the bacterial host cell.

The present invention is directed to a method for producing achondroitin, comprising culturing a non-pathogenic bacterial host cellcomprising any of the constructs of the invention under fermentationconditions sufficient for production of the chondroitin.

The present invention is directed to a method for producing anon-pathogenic bacterial host cell comprising transferring any of theconstructs of the invention to a non-pathogenic bacterial host cell.

In some embodiments, a gene cluster or gene of any of the constructs ofthe invention is integrated into a chromosome of the bacterial host cellin any of the methods of the invention.

In some embodiments, two or more copies of a gene cluster or gene of anyof the constructs of the invention are integrated into a chromosome ofthe bacterial host cell in any of the methods of the invention. In someembodiments, the two or more copies of the gene cluster or gene includetwo or more copies of the same gene or gene cluster.

In some embodiments, the chondroitin produced by any of the methods ofthe invention is non-fructosylated.

In some embodiments, the methods of the present invention furthercomprise sulfating the chondroitin.

The present invention is directed to a method for producing achondroitin sulfate, comprising producing a chondroitin by any of themethods of the invention; and sulfating the chondroitin.

In some embodiments, the process of sulfating the chondroitin in any ofthe methods of the invention comprises mixingsulfurtrioxide-triethylamine complex or chlorosulfonic acid with thechondroitin in formamide.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is or is derived from a non-pathogenic organism selectedfrom the group consisting of Escherichia, Pseudomonas, Xanthomonas,Methylomonas, Acinetobacter, and Sphingomonas.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is or is derived from a gram-negative organism.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is a Xanthomonas campestris. In some embodiments, the X.campestris is a bacterial strain selected from the group consisting ofMSC255, MSC256, MSC257, MSC225, and MSC226.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is a non-pathogenic E. coli. In some embodiments, thenon-pathogenic E. coli is selected from the group consisting of E. coliK-12 and E. coli B. In some embodiments, the E. coli K-12 is a bacterialstrain selected from the group consisting of MSC188 and MSC175. In someembodiments, the E. coli B is bacterial strain MSC364.

In some embodiments, an endogenous gene of the bacterial host cell inany of the methods of the invention is deleted or inactivated byhomologous recombination.

In some embodiments, the bacterial host cell in any of the methods ofthe invention does not express extracellular polysaccharides endogenousto the host cell.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is suitable for conjugal transfer from a laboratorycloning strain.

In some embodiments, the methods of the invention further compriserecovering the chondroitin from the bacterial host cell.

In some embodiments, the methods of the invention further compriserecovering the chondroitin from extracellular culture medium.

In some embodiments, 1 g/L to 50 g/L of the chondroitin is secreted fromthe bacterial host cell in any of the methods of the invention in 24 to72 hours. In some embodiments, 5 g/L to 50 g/L of the chondroitin issecreted from the bacterial host cell in 24 to 72 hours. In someembodiments, 15 g/L to 50 g/L of the chondroitin is secreted from thebacterial host cell in 24 to 72 hours.

In some embodiments, any of the methods of the invention furthercomprise purifying the chondroitin.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is cultured at 25° C. to 37° C.

In some embodiments, the bacterial host cell in any of the methods ofthe invention is cultured in a medium comprising glycerine.

The present invention is directed to the chondroitin produced by any ofthe methods of the invention.

The present invention is directed to a composition comprising thechondroitin produced by any of the methods of the invention.

The present invention is directed to an antibody or antibody fragmentthat binds to an amino acid sequence selected from the group of: SEQ IDNO:92 of KpsF, SEQ ID NO:93 of KpsE, SEQ ID NO:94 of KpsD, SEQ ID NO:95of KpsU, SEQ ID NO:96 of KpsC, SEQ ID NO:97 of KpsS. SEQ ID NO:91 ofKpsT, SEQ ID NO:83 of KfoA, SEQ ID NO:84 of KfoB, SEQ ID NO:85 of KfoC,SEQ ID NO:86 of KfoI (Orf3), SEQ ID NO:87 of KfoE, SEQ ID NO:88 of KfoH(Orf1), SEQ ID NO:89 of KfoF, and SEQ ID NO:90 of KfoG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of chondroitin and chondroitin sulfate. FIG.1B shows the linkage between chondroitin sulfate and core protein ofproteoglycans.

FIG. 2 shows the organization of the gene cluster involved in thesynthesis of the E. coli K4 capsule, as proposed prior to thisinvention. The organization of region 2 shown in this figure is asdescribed in Ninomiya et al. (J. Biol. Chem. 2002; 277:21567-21575).Regions 1 and 3 of the E. coli K4 capsule gene cluster were notsequenced prior to this invention and therefore, their structure was notknown prior to this invention.

FIG. 3 shows the analysis by the present inventors of the E. coli K4capsule region 2 sequence (AB079602) as described in Ninomiya et al. (J.Biol. Chem. 2002; 277:21567-21575). FIG. 3A shows the presence ofadditional putative coding sequences orf1, orf2 and orf3, and furthershows that based on sequence alignment of region 2 from E. coli K4 withP. multocida serotypes B and E; genes (alignment data shown in FIG. 3B),kfoD, orf3, kfoE, orf1, from E. coli K4 have homologs among the P.multocida M1404 serotype B genes hcbDEFG and the P. mulocida P1234serotype E genes echDEFG. Homologs are connected by two-headed arrows.

FIG. 4 relates to the sequences of the region 2 genes of E. coli K4strain ATCC 23502 as determined by the present inventors. FIG. 4A listsdifferences between the sequences determined by the present inventors ascompared to the sequences previously reported by Ninomiya et al. FIG. 4Bshows a comparison of the predicted amino acid sequences encoded by thesequences of the region 2 genes, as determined by the present inventors(shown in FIG. 4B as SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:26; SEQ IDNO:24; and SEQ ID NO:20) with those encoded by the sequences reported byNinomiya et al. (shown in FIG. 4B as K4 Kfo putORF2 and K4KfoG_BAC00518; K4 Kfo putORF_(—)1; K4 KfoE_BAC00520; K4 KfoD_BAC00521;K4 KfoB_BAC00524).

FIG. 5 shows the organization of the K4 capsule gene cluster from the E.coli K4 strain U1-41. The gene cluster contains 17 open reading frames(exclusive of IS2) that are predicted to encode proteins.

FIG. 6 diagrammatically represents the structure of synthetic genesconstructed as three segments, kpsFEDUCS (the “FS segment”),kpsMTkfoABCFG (the “MG segment”) and kfoDIEH (the “DH segment”). Asdepicted, restriction sites were incorporated at strategic locations toallow assembly of the synthetic fragments into one or more operons andto facilitate manipulation of individual genes.

FIG. 7A shows the pop in, pop-out strategy for constructing derivativebacterial strains by deletion of a particular gene or gene cluster. FIG.7B shows the map of the suicide vector pCX027 (SEQ ID NO:141) used toemploy this strategy in Xanthomonas campestris in this strategy.

FIGS. 8A-8U represent the DNA maps for plasmids and DNA fragments pBHR1,pDD39, pDD42, pDD47, pREZ6, pDD49, pJ201:11352, pDD50, pDD54,pJ241:10662, pJ241:10664, pJ241:10663, pDD37, pDD38, pDD51, pDD52,pDD57, pDD58, pDD61, pDD62, pDD63, pDD59, pDD67, pDD60, pDD66, pBR1052,pMAK-CL, pDD74, pDD76, pDD73, pDD77, pDD79, pDD80, pCX045, pCS048,pCX039, pCX044, pCX040, pCX042, pCX041, pCX043, MSC467, MSC561, andpBR1087 of the present invention.

FIG. 9 shows examples of results from western blots performed usingantisera directed against proteins encoded by the E. coli K4 group 2capsule gene cluster.

FIGS. 10A-10D show the DNA maps of plasmid constructs used to introducecloned E. coli K4 chondroitin biosynthesis genes into Xanthomonascampestris.

FIG. 11A shows a typical calibration curve for the K4 fructosylatedchondroitin capsular polysaccharide (“K4P”) measured in inhibitoryELISA. FIG. 11B shows a typical standard curve of the disaccharide,2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronicacid)-D-galactose (“Δdi-0S”), measured in the chondroitinase/HPLC assayfor chondroitin.

FIG. 12 shows chondroitinase digestibility of recombinant chondroitin.

FIG. 13 shows chondroitinase digestibility of both K4 fructosylatedchondroitin capsular polysaccharide (K4P) and defructosylated K4P(DFK4P).

FIGS. 14A-14X show the DNA maps for plasmid constructs pCX096, pCX097,pCX100, pCX101, pCS102, pCX075, pCX082, pCX081, pCX092, pBR1077,pBR1082, pCX050, pCX070, pCX093, pCX094, pCX095, pMAK705pl, pBR1103,pBR0193lacZ, pBR100-lac, pBR1094mtl, pBR101-mtl, pBR1095fru, andpBR1102-fru of the present invention.

FIG. 15 shows a family tree of chondroitin-producing E. coli strains andthe steps used for strain derivation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to constructs and recombinant cellsfor the production of chondroitin, methods of producing chondroitin,chondroitin produced from the methods, and uses of the chondroitin. Asdescribed herein, the present invention is based on a novel technologythat allows production of chondroitin and chondroitin sulfate. Thepresent invention meets an important need in the art by providing asafe, consistent, and reliable supply of chondroitin and chondroitinsulfate at lower cost, while providing superior product quality. Thisprocess can also provide a vegetarian and kosher product. Therecombinantly produced chondroitin can be sulfated using known methodsto form chondroitin sulfate. Accordingly, the present invention includesmethods of sulfation of the recombinantly produced chondroitin, therecombinantly produced chondroitin sulfate product, as well as uses ofthe recombinantly produced chondroitin sulfate product.

As described in detail below, the present inventors sequenced the E.coli K4 gene cluster encoding proteins involved in biosynthesis of theE. coli K4 fructosylated chondroitin capsular polysaccharide (K4P),synthesized and assembled DNA segments based on the native sequence,transferred these genes into alternative host cells that are suitablefor large scale fermentation, and demonstrated production of recombinantfructosylated chondroitin capsular polysaccharide in these host cells.Since it is preferable that the alternative host cells should producenon-fructosylated chondroitin, genes responsible for fructosylation ofchondroitin by E. coli K4 were identified and deleted from the E. coliK4 chondroitin biosynthesis gene set transferred to the alternativehosts. As a result, the alternative hosts containing this gene setproduced non-fructosylated chondroitin. This recombinant chondroitin(rCH) produced by the alternative host can be sulfated to produce achondroitin sulfate product.

As used herein, the term “K4P” refers to the native or naturallyoccurring fructosylated chondroitin capsular polysaccharide assynthesized by the wild type E. coli K4 strain. The term “chondroitin”refers to the chondroitin backbone. Chondroitin can be fructosylated orunfructosylated (or non-fructosylated). The term “chondroitin,” as usedherein, includes both fructosylated and unfructosylated forms, unlessspecifically noted. Furthermore, as used herein, the term chondroitinrefers to unsulfated chondroitin. The chondroitin produced by themethods of present invention can be sulfated by enzymatic or chemicalmeans, as explained in detail below, in which case it is referred to aschondroitin sulfate.

In one aspect, the present invention comprises DNA constructs comprisingthe E. coli K4 gene sets or gene clusters. The term “K4 gene cluster” asused herein refers to the set of genes from E. coli K4 that are involvedin the biosynthesis of the chondroitin-like capsular polysaccharide(K4P). The term “K4 gene cluster” can refer to all the genes from E.coli K4 that are involved in the biosynthesis of the chondroitin-likecapsular polysaccharide or to a subset of these genes.

As described in detail in Example 1, E. coli K4 contains a set ofmultiple genes that are involved in the synthesis of a chondroitin-likecapsular polysaccharide referred to as K4P. As noted above, thispolysaccharide consists of a chondroitin backbone that is modified bythe addition of a fructose residue. As shown in FIG. 2, these genes areorganized in three main regions (Region 1 (“R1”), Region 2 (“R2”), andRegion 3 (“R3”)). Based on the sequence of region 2 described byNinomiya et al. (2002) (GenBank accession no. AB079602), region 2 waspredicted to comprise seven genes relating to capsule biosynthesis,kfoA, kfoB, kfoC, kfoD, kfoE, kfoF and kfoG. Ninomiya et al. did notdisclose the sequences of the expected region 1 and region 3 portions ofthe E. coli K4 capsule gene cluster. However, based on the knownorganization of other E. coli capsule gene clusters, region 1 would bepredicted to comprise six genes, kpsF, kpsE, kpsD, kpsU, kpsC, kpsS andregion 3 would be predicted to comprise two genes, kpsM and kpsT. ThekpsM, kpsT, kpsD, kpsE, kpsC and kpsS genes encode proteins required fortranslocation of the capsular polysaccharide from the cell cytoplasm tothe cell surface where the mature capsular polysaccharide is believed tobe anchored to the outer cell membrane through a covalent linkage to alipid component of that membrane (Whitfield, 2006). The kpsF and kpsUgenes encode proteins that are predicted to catalyze steps in thebiosynthesis of CMP-Kdo. A role of CMP-Kdo in the biosynthesis ofchondroitin capsules in E. coli has been proposed (Roberts, Annu. Rev.Microbiol. 1996; 50:285-315) but has not been demonstratedexperimentally (Whitfield, 2006) Thus, prior to the disclosure of thepresent invention the entire K4 gene cluster was thought to comprise 15genes.

In order to confirm the sequence reported by Ninomiya et al. (GenBankaccession no. AB079602), the present inventors sequenced region 2 of theK4 capsule gene cluster from the E. coli K4 strain ATCC 23502. When thesequence determined by the present inventors and the AB079602 sequencewere compared, single base pair differences were discovered, includingsubstitutions, deletions and insertions at 26 positions. As explained indetail in Example 1, some of these differences result in substantialdifferences in the predicted amino acid sequences of the region 2proteins encoded by the gene cluster. Furthermore, the present inventorsexamined the regions identified by Ninomiya et al. as intergenicsequences separating the genes and identified three additional openreading frames orf1 (also referred to herein as kfoH), orf2 and orf3(also referred to herein as kfoI) in region 2 that were previouslyunidentified.

In order to determine the correct sequence of the entire K4 gene clustercomprising genes from all three regions. E. coli serotype K4 strainU1-41 was obtained from the Statens Serum Institut (Copenhagen,Denmark). U1-41 is the progenitor of the ATCC 23502 strain and has beenreported to produce K4 capsular polysaccharide in culture. It is alsothe K4 reference strain for E. coli serotyping and was used to producethe polysaccharide preparation used for K4P structural determination byRodriguez et al. (1988). A total of approximately 23 kb of DNA spanningthe K4 capsule gene cluster in E. coli U1-41 was sequenced. Thissequence (SEQ ID NO:117) confirmed the presence of kpsF, kpsE, kpD,kpsD, kpsC and kpsS genes in region 1 and the presence of kpsM and kpsTgenes in region 3. The region 2 sequence of U1-41 and the sequencedetermined by the present inventors for region 2 of ATCC 23502 werefound to be identical.

As described in detail in Example 1, the gene cluster from U1-41 wasfound to contain (exclusive of IS2 sequences) 17 open reading frames,(instead of 15 as previously described by Ninomiya et al.) that arepredicted to encode proteins related to biosynthesis of chondroitin. Thearrangement of these genes is typical for an E. coli group 2 capsulegene cluster. Region 1, comprising conserved genes kpsF, kpsE, kpsD,kpsU, kpsC and kpsS, and region 3, comprising conserved genes kpsM andkpsT, flank the 9 open reading frames of region 2. Region 1 and region 3genes include proteins that are required for synthesis and translocationof all group 2 capsules in E. coli. Region 1 also includes two genes(kpsF and kpsU) that encode enzymes that are, as noted above, predictedto catalyze steps in the biosynthesis of CMP-Kdo. Of the nine genesidentified in region 2, three encode proteins with clearly definedactivities relating to capsule biosynthesis: kfoA (UDP-GilcNAcepimerase, which converts UDP-GlcNAc to the UDP-GalNAc precursor), kfoF(UDP-Glc dehydrogenase, which converts UDP-Glc to the UDP-GlcUAprecursor) and kfoC (chondroitin synthase, i.e. the polymerase, whichcan add either one of the precursors, UDP-GalNAc or UDP-GlcUA, to anacceptor chondroitin molecule).

Functions of proteins encoded by other genes present in region 2 of theK4 capsule gene cluster, kfoB, kfoG, kfoD, kfoE, kfoH (orf1) and kfoI(orf3), were not known. The kfoB and kfoG genes encode proteins that arehomologous to those encoded by genes present in capsule clusters ofbacteria known to produce other glycosaminoglycan (GAG) capsules such asP. multocida serotypes A. F and D (Townsend et al., J. Clin. Microbiol.2001:39:924-929) and E. coli serotype K5 (Petit et al., Mol. Microbiol.1995; 4:611-620). This circumstantial evidence suggested that kfoB andkfoG can play a role in biosynthesis of the GAG-containing K4 capsule.As explained in detail in Example 7, the present inventors have foundthat kfoB and kfoG genes are not essential for the production ofchondroitin in E. coli, but that kfoG gene is required for the optimalproduction of chondroitin.

Prior to the present invention, no evidence implicated kfoD, kfoE, kfoH(or orf1) and kfoI (or orf3) as being involved in the biosynthesis ofthe K4 capsule. Interestingly, the four contiguous K4 genes, kfoD, kfoI(or orf3), kfoE, and kfoH (or orf1), were found to have homologs amongthe contiguous P. multocida serotype B genes hcbDEFG and the P.multocida serotype E genes echDEFG. However, these two Pasteurellastrains are not known to be chondroitin producers and the role of thesegenes in E. coli K4 was not known prior to the present invention.Therefore, it appeared that kfoD, kfoI (orf3), kfoE and kfoH (orf1)might not be involved in the synthesis of chondroitin. As shown inExamples 6 and 7, none of these genes are required for biosynthesis ofchondroitin, but one or more of these genes are essential forfructosylation of the chondroitin produced by the K4 gene set.

Using the sequence of the U1-41 K4 capsule gene cluster as the basis,the present inventors further designed synthetic genes that arecodon-optimized for expression in hosts such as E. coli, Xanthomonascampestris, Sphingomonas elodea and Bacillus subtilis. The design andsynthesis of these coxion-optimized genes is explained in detail inExample 2. Example 4 describes the construction of plasmid vectors forthe expression of these genes in heterologous bacteria.

The complete nucleotide sequences of the codon-optimized genes of thepresent invention, and the amino acid sequences encoded by them, are asfollows. The complete nucleotide sequence for kpsF used in the presentinvention is represented herein as SEQ ID NO: 1. kpsF is a 981nucleotide sequence (not including the stop codon) which encodes a 327amino acid sequence, represented herein as SEQ ID NO:2. The completenucleotide sequence for kpsE is represented herein as SEQ ID NO:3. kpsEis a 1146 nucleotide sequence (not including the stop codon) whichencodes a 382 amino acid sequence, represented herein as SEQ ID NO:4.The complete nucleotide sequence for kpsD is represented herein as SEQID NO:5. kpsD is a 1674 nucleotide sequence (not including the stopcodon) which encodes a 558 amino acid sequence, represented herein asSEQ ID NO: 6. The complete nucleotide sequence for kpsU is representedherein as SEQ ID NO:7. kpsU is a 738 nucleotide sequence (not includingthe stop codon) which encodes a 246 amino acid sequence, representedherein as SEQ ID NO:8. The complete nucleotide sequence for kpsC isrepresented herein as SEQ ID NO:9. kpsC is a 2025 nucleotide sequence(not including the stop codon) which encodes a 675 amino acid sequence,represented herein as SEQ ID NO:10. The complete nucleotide sequence forkpsS is represented herein as SEQ ID NO: 11. kpsS is a 1209 nucleotidesequence (not including the stop codon) which encodes a 403 amino acidsequence, represented herein as SEQ ID NO:12. The complete nucleotidesequence for kpsM is represented herein as SEQ ID NO:13. kpsM is a 774nucleotide sequence (not including the stop codon) which encodes a 258amino acid sequence, represented herein as SEQ ID NO:14. The completenucleotide sequence for kpsT is represented herein as SEQ ID NO:15. kpsTis a 666 nucleotide sequence (not including the stop codon) whichencodes a 222 amino acid sequence, represented herein as SEQ ID NO:16.The complete nucleotide sequence for kfoA is represented herein as SEQID NO:17. kfoA is a 1017 nucleotide sequence (not including the stopcodon) which encodes a 339 amino acid sequence, represented herein asSEQ ID NO:18. The complete nucleotide sequence for kfoB is representedherein as SEQ ID NO:19. kfoB is a 1638 nucleotide sequence (notincluding the stop codon) which encodes a 546 amino acid sequence,represented herein as SEQ ID NO:20. The complete nucleotide sequence forkfoC is represented herein as SEQ ID NO:21. kfoC is a 2058 nucleotidesequence (not including the stop codon) which encodes a 686 amino acidsequence, represented herein as SEQ ID NO:22. The complete nucleotidesequence for kfoD is represented herein as SEQ ID NO:23. kfoD is a 1431nucleotide sequence (not including the stop codon) which encodes a 477amino acid sequence, represented herein as SEQ ID NO:24. The completenucleotide sequence for kfoE is represented herein as SEQ ID NO:25. kfoEis a 1566 nucleotide sequence (not including the stop codon) whichencodes a 522 amino acid sequence, represented herein as SEQ ID NO:26.The complete nucleotide sequence for kfoF is represented herein as SEQID NO:27. kfoF is a 1167 nucleotide sequence (not including the stopcodon) which encodes a 389 amino acid sequence, represented herein asSEQ ID NO:28. The complete nucleotide sequence for kfoG is representedherein as SEQ ID NO:29. kfoG is a 1464 nucleotide sequence (notincluding the stop codon) which encodes a 488 amino acid sequence,represented herein as SEQ ID NO:30.

The complete nucleotide sequence for orf1 (also referred to herein askfoH) is represented herein as SEQ ID NO:31. orf1 is a 723 nucleotidesequence (not including the stop codon) which encodes a 241 amino acidsequence, represented herein as SEQ ID NO:32. The complete nucleotidesequence for orf3 (also referred to herein as kfoI) is representedherein as SEQ ID NO:33. orf3 is a 378 nucleotide sequence (not includingthe stop codon) which encodes a 126 amino acid sequence, representedherein as SEQ ID NO:34.

In various embodiments, the present invention comprises DNA constructscomprising the E. coli K4 gene cluster, one or more regions of the E.coli K4 gene cluster, one or more subsets of genes from the E. coli K4gene cluster, one or more individual genes from the E. coli K4 genecluster, or combinations thereof, wherein the constructs are useful forthe purpose of producing chondroitin or increasing the amount ofchondroitin in a bacterial host cell. In various embodiments, theconstructs can include the entire 17 gene cluster described above or oneor more genes of the 17 gene cluster described above, i.e. kpsF, kpsE,kpsD, kpsU. kpsC, kpsS, kpsM, kpsT, kfoA, kfoB, kfoC, kfoD, kfoE, kfoF,kfoG, kfoH and kfoI. In some embodiments, the construct comprises one ormore regions of the K4 cluster (i.e., regions 1, 2, and/or 3 asdescribed herein). In some embodiments, the construct comprises one ormore subsets of genes from the K4 cluster (including subsets of genesfrom regions 1, 2, and/or 3 as described herein). The construct cancomprise a gene cluster in which a gene in the cluster is present in anyorder relative to any other gene in the cluster. As such, the orderingof genes in a gene cluster within the construct can be different fromthe naturally occurring order of genes within the K4 cluster. Similarly,the construct can comprise a region, a subset of genes, or a gene fromthe K4 cluster that can be in any order within the construct relative toany other region, subset of genes, or individual genes from the K4cluster. In some embodiments, the genes are present in a specified orderin the construct. The constructs can include one or more native genes(i.e., the genes having the sequence present in E. coli K4 U1-41 orother serotype K4 strains) that were isolated from the E. coli serotypeK4 strain U1-41 mentioned above and/or one or more synthetic genes,i.e., the genes based on the native genes isolated from U1-41 but inwhich the DNA sequences have been modified for optimal codon usage inthe bacterial host cell, without altering the amino acid sequencesencoded by those genes. The design and preparation of such syntheticgenes is explained in Example 2.

As noted above and further explained in detail in Examples 6 and 7, oneor more of the kfoD, kfoI, kfoE and kfoH genes are essential forfructosylation of chondroitin in E. coli, but none of these genes isrequired for synthesis of chondroitin. Simultaneous omission orinactivation of all four of these genes results in the production ofunfructosylated chondroitin. In some embodiments, a construct of theinvention does not contain a functional gene of one or more of kfoD.kfoI, kfoE and kfoH. In other words, a functional gene of one or more ofkfoD, kfoI, kfoE and kfoH is absent from the construct in theseembodiments. A construct that does not contain a functional gene (i.e.,a construct in which a functional gene is absent) includes constructs inwhich the entire gene is absent as well as constructs in which the geneor portion thereof is present but is non-functional (i.e., inactive). Insome embodiments, a construct of the invention comprises a gene clusterthat has been modified to inactivate one or more of kfoD, kfoI, kfoE andkfoH.

In some embodiments, the present invention includes a construct thatcomprises a gene cluster comprising kpsF, kpsE, kpsD, kpsU, kpsC. kpsS,kfoA, kfoC, and kfoF, wherein the construct does not contain afunctional gene of one or more of kfoD, kfoI, kfoE and kfoH. In someembodiments, the construct is suitable for producing a chondroitin in anon-pathogenic bacterial host cell as described herein. In someembodiments, the chondroitin is non-fructosylated. In some embodiments,the construct can further comprise kfoG and/or kfoB. As noted above,kfoB and kfoG genes were not found to be essential for the production ofchondroitin, but the kfoG gene was found to be required for the optimalproduction of chondroitin (See example 7). In some embodiments, theconstruct of the present invention can further comprise kpsM and/orkpsT.

In some embodiments, the constructs are useful for the production ofrecombinant chondroitin that is secreted from the cell.

In some embodiments, these constructs include the expression vectorspDD66 (expression vector containing kpsMT-kfoABCFG-kpsFEDUCS), pDD67(expression vector containing kpsFEDUCS-kpsMT-kfoABCFG), pCX040(expression vector containing kpsMT-kfoACFG-kpsFEDUCS), pCX041(expression vector containing kpsMT-kfoABCF-AksFEDUCS), pCX042(expression vector containing kpsFEDUCS-kpsMT-kfoACFG), pCX043(expression vector containing kpsFEDUCS-kpsMT-kfoABCF), and pCX096(expression vector containing kpsFEDUCS-kfoABCFG). Another embodiment isexpression plasmid pBR1052. As described in Example 4, pBR1052 containsthe same K4 gene set as pDD66 (kpsMT-kfoABCFG-kpsFEDUCS) andadditionally has a second copy of the Pm promoter sequence insertedimmediately upstream of the kpsF gene. The nucleotide sequence of pDD66is denoted by SEQ ID NO:35; the nucleotide sequence of pDD67 is denotedby SEQ ID NO:36; the nucleotide sequence of pCX040 is denoted by SEQ IDNO:37; the nucleotide sequence of pCX041 is denoted by SEQ ID NO:38; thenucleotide sequence of pCX042 is denoted by SEQ ID NO:39; the nucleotidesequence of pCX043 is denoted by SEQ ID NO:40; the nucleotide sequenceof pCX096 is denoted by SEQ ID NO:149; and the nucleotide sequence ofpBR1052 is denoted by SEQ ID NO:41. The design and construction of theseDNA constructs is explained in detail in Example 4.

In some embodiments, the present invention includes constructs that areuseful for the purpose of producing intracellular chondroitin, i.e.,chondroitin that is not secreted from the host cell. Intracellularproduction of chondroitin can be desirable in order to eliminateviscosity of the fermentation resulting from high levels ofpolysaccharide in the culture medium. In addition, it is possible thatintracellular production could achieve higher levels of chondroitin thansecretion. In some embodiments, the construct does not contain afunctional gene of at least one of kpsM and kpsT of region 3. In someembodiments, the construct comprises a gene cluster that does notcontain or has been modified to inactivate at least one of kpsM and kpsTof region 3. In some embodiments, the construct does not contain afunctional gene of at least one of kpvE, kpsD, kpsC and kpsS ofregion 1. In some embodiments, the construct comprises a gene clusterthat does not contain or has been modified to inactivate at least one ofkpsE, kpsD, kpsC and kpsS of region 1. In some embodiments, theconstruct does not contain a functional gene of at least one of kpsM andkpsT of region 3 and at least one of kpsE, kpsD, kpsC and kpsS ofregion 1. In some embodiments, the construct comprises a gene clusterthat does not contain or has been modified to inactivate at least one ofkpsM and kpsT of region 3 and at least one of kpsE, kpsD, kpsC and kpsSof region 1. These constructs are described in Examples 4 and 9.

In some embodiments, the present invention includes a constructcomprising a gene cluster comprising kfoA, kfoC, and kfoF, wherein thegene cluster does not contain a functional gene of one or more of kpsM,kpsT, kpsE, kpsD, kpsC and kpsS. In some embodiments, the construct issuitable for producing a chondroitin in a non-pathogenic bacterial hostcell as described herein. In some embodiments, the chondroitin is notsecreted from the host cell. In some embodiments, the chondroitin isnon-fructosylated. In some embodiments, the construct also does notcontain a functional gene of one or more of kfoD, orf3, kfoE, and orf1.In some embodiments, the construct can further comprise kfoG and/orkfoB. In some embodiments, the construct comprises kfoA, kfoB, kfoC,kfoF and kfoG.

In some embodiments, a construct of the present invention includes agene selected from the group consisting of kfoA, kfoB, kfoC, kfoF, kfoG,and a combination thereof, wherein the construct does not contain afunctional gene of one or more of kpsM, kpsT, kpsE, kpsD, kpsC and kpsS.In some embodiments, the construct is suitable for producing achondroitin in a non-pathogenic bacterial host cell as described herein.In some embodiments, the construct is suitable for increasing the amountof a chondroitin in a non-pathogenic bacterial host cell as describedherein. In some embodiments, the construct is transferred to a bacterialhost cell comprising one or more existing copies of the E. coli K4 genecluster, regions of the cluster, subsets of genes of the cluster, orgenes of the cluster that have been integrated into the host chromosome.In some embodiments, the chondroitin is non-fructosylated. In someembodiments, the construct also does not contain a functional gene ofone or more of kfoD, orf3, kfoE, and orf1. In some embodiments, theconstruct comprises kfoA, kfoB, kfoC, kfoF and kfoG.

In some embodiments, a construct of the present invention comprises theexpression vectors pCX039 (expression vector containing kfoABCFG),pCX044 (expression vector containing kfoACFG), pCX092 (expression vectorcontaining kfoABCF), pCX045 (expression vector containingkpsMT-kfoABCFG-kpsFEDUS), and pCX048 (expression vector containingkpsM-kfoABCFG-kpsFEDUCS). The nucleotide sequence of pCX039 is denotedby SEQ ID NO:42; the nucleotide sequence of pCX044 is denoted by SEQ IDNO:43; the nucleotide sequence of pCX092 is denoted by SEQ ID NO:154;the nucleotide sequence of pCX045 is denoted by SEQ ID NO:44; and thenucleotide sequence of pCX048 is denoted by SEQ ID NO:45. The design andconstruction of these DNA constructs is explained in detail in Example4.

In some embodiments, a construct of the present invention comprises theexpression vectors pCX075 (expression vector containing kfoABFG), pCX081(expression vector containing kfoABCG), pCX082 (expression vectorcontaining kfoBCFG), pCX101 (expression vector containingkfoABCFG-kpsMT), pBR1102 (expression vector containing kfoABCFG),pBR1100 (expression vector containing kfoABCFG), and pBR1101 (expressionvector containing kfoABCFG). The nucleotide sequence of pCX075 isdenoted by SEQ ID NO:153; the nucleotide sequence of pCX081 is denotedby SEQ ID NO:151; the nucleotide sequence of pCX082 is denoted by SEQ IDNO:152; the nucleotide sequence of pCX101 is denoted by SEQ ID NO:150;the nucleotide sequence of pBR1102 is denoted by SEQ ID NO:170; thenucleotide sequence of pBR1100 is denoted by SEQ ID NO:171; and thenucleotide sequence of pBR1101 is denoted by SEQ ID NO: 172. The designand construction of these DNA constructs is explained in detail inExamples 18 and 20.

The constructs of the present invention can comprise one or more genesthat are modified for optimal codon usage in a bacterial host cell asdescribed herein.

The constructs of the present invention can further comprise a promoter.The promoter should be capable of driving expression of the gene clusterin a bacterial host cell as described herein. Numerous such promoters,which are useful to drive expression in the desired bacterial host cellare familiar to those skilled in the art and can be used in the presentinvention. Examples of promoters that have been commonly used to expressheterologous proteins include, without limitation, Pin, lac, trp, lac,λpL, T7, phoA, araC, xapA, cad and recA (See, e.g., Weikert et al.,Curr. Opin. Biotechnol. 1996; 7:494-499). Such promoters can beconstitutive or inducible. Termination control regions can also bederived from various genes native to the preferred hosts. Optionally, atermination site may be unnecessary.

In some embodiments, the constructs of the present invention comprisethe Pm promoter along with the xylS regulatory gene (Mermod et al., J.Bacteriol. 1986:167:447-54). The Pm promoter, which is isolated fromPseudomonas putida TOL plasmid and its regulatory gene xylS provide astrong, well regulated promoter shown to function in a variety of gramnegative bacteria (Blatny et al., Plasmid 1997; 38:35-51). The XylSprotein can exist as a monomer or dimer. In the dimeric form, the XylSprotein can bind to the Pm promoter and stimulate transcription.Dimerization of the XylS protein, and thus transcriptional initiation atthe Pm promoter, is enhanced by certain effector molecules such asmeta-toluic acid (3-methylbenzoate) which bind directly to XylS andpromote dimerization of the protein. (Dominguez-Cuevas et al., J. Bact.2008; 190:3118-3128). The promoter can be operably linked to one or moregenes of the gene cluster.

The constructs of the present invention can further comprise a secondpromoter. For example, if analysis of expression of cloned K4 genes inan alternative host indicated that the level of expression of a certaingene, or gene set, is less than optimal, a second promoter can be addedto the expression construct at a location that is selected so as toenhance the transcription of the gene or gene set that is not expressedat an optimal level. Typically, the added promoter would be insertedimmediately upstream (i.e., 5′ to) the gene or gene set of interest. Thesecond promoter can be Pm, or any of the promoters listed above asexamples of promoters useful expressing K4 gene sets. In someembodiments, the second promoter can be Pm. The second promoter can beoperably linked to one or more genes of the gene cluster. In oneembodiment, the second promoter can be operably linked to kpsFEDUCS geneset. See, for instance, expression vector pBR1052 as described inExample 4. The genes, or combinations of genes, that it would beadvantageous to express, or augment the expression of, by use of asecond promoter can be determined empirically for any given plasmid, orchromosomal, gene set by western blot analysis.

The constructs of the present invention can further comprise anantibiotic resistance gene that confers resistance to a particularantibiotic. Such genes are well known in the art. Examples of antibioticresistance genes include, without limitation, chloramphenicol resistancegene (CamR), kanamycin resistance gene (KanR), ampicillin resistancegene (AmpR), tetracycline resistance gene (TetR), spectinomycinresistance gene (SpcR), sulfonamide resistance gene (SuR), bleomycinresistance gene (BleR), streptomycin resistance gene (StrR),carbenicillin resistance gene (CbR) and erythromycin resistance gene(EryR).

The constructs of the present invention are useful for producingchondroitin in a bacterial host cell. While any bacterial cell can beused as a host cell in the present invention, in some embodiments thehost is a gram-negative bacterium. Examples of gram negative bacteriainclude, without limitation, Escherichia, Pseudomonas, Xanthonmonas,Methylomonas, Acinetobacter and Sphingomonas. In some embodiments thehost is a non-pathogenic gram-negative bacterium. Examples ofnon-pathogenic gram-negative bacteria include without limitation,non-pathogenic E. coli such as E. coli K-12 or E. coli B, Xanthomonascampestris, Sphingomonas elodea and Pseudomonas putida.

In some embodiments, an endogenous gene of a bacterial host cell asdescribed herein is deleted or inactivated by homologous recombination.

Derivatives of hosts that are unable to manufacture their nativeextracellular polysaccharides are desirable. Use of such derivativehosts would facilitate the visual and chemical identification ofbiosynthesis of recombinant chondroitin (rCH), as well as thepurification of the rCH produced by the host when a K4 gene set isintroduced. Furthermore, biosynthesis of rCH in appropriately designedderivative hosts would not be limited by competition with the nativepolysaccharide synthesis. For example, inactivation or deletion of thefirst glycosyltransferase genes of a native polysaccharide biosyntheticpathway would prevent utilization of any potential lipid carrier by thenative pathway and prevent competition between the enzymes of the nativepathway and the K4 enzymes for the lipid carrier, or any other cellularcomponent(s) that acts on nascent polysaccharide chains and might belimiting in availability. Inactivation (e.g. by deletion) of the entirenative biosynthetic gene clusters would remove most of the competitiveelements, but could potentially have undesirable effects on physiologyand/or membrane structure.

As described in detail in Example 3, the present inventors have, by wayof example and without intending to be limited to these examples, usedE. coli K-12 (“K-12”), E. coli B (“EcB”), and Xanthomonas campestris pv.campestris (“Xcc”) as hosts for the expression of the constructs of thepresent invention. Generation of derivative hosts that comprisedeletions in one or more genes of the gene cluster that encode enzymesfor synthesis of native extracellular polysaccharides was carried outusing a two-step, “pop-in/pop-out” homology-driven method as describedin detail in Example 3. For example, colanic acid (M antigen) is anextracellular polysaccharide produced by many enteric bacteria. E. coliK-12 and E. coli B strains that are deficient or defective in thecolanic acid biosynthesis were created. Strains MSC88 and MSC175 arederivatives of E. coli K-12 strain that comprise a deletion of theentire colanic acid operon, and the wcaJ gene encoding theglycosyltransferase enzyme responsible for loading of the first sugaronto a lipid carrier during colanic acid biosynthesis, respectively.Strain MSC364 is a derivative of E. coli B that comprises a deletion ofthe entire colanic acid operon. Similarly, Xanthomonas campestris pv.campestris strains that are deficient or defective in the biosynthesisof the extracellular polysaccharide xanthan gum were created. StrainsMSC225 and MSC226 are derivatives of Xcc strain that comprise a deletionof the gumD gene encoding the glycosyltransferase I enzyme and strainsMSC255, MSC256, and MSC257 comprise a deletion of the entire xanthan gumoperon.

The present invention is directed to a method for producing anon-pathogenic bacterial host cell comprising any one or more of theconstructs of the present invention, comprising transferring any one ormore of the constructs of the present invention to a non-pathogenicbacterial host cell. The constructs of the present invention can beintroduced into bacterial host cells by any known method for expressionof the genes present within the constructs. Such methods can include,without limitation, transformation, electroporation, conjugation ortransduction.

The present invention is directed to a non-pathogenic bacterial hostcell comprising any one or more of the constructs of the presentinvention. As such, the present invention also includes various strainsthat were created by introducing the constructs comprising expressionvectors of the present invention into the host strains, including thederivative strains comprising deletions. Certain examples are describedin detail in Examples 6-9, 11, 13 and 14.

In some embodiments, the genes contained within the constructs of theinvention are introduced into the chromosome of the recipient hoststrain such that the genes are integrated within the host chromosome.Placing the cloned genes in the chromosome provides the advantage ofeliminating the requirement for maintaining selective pressure tomaintain the plasmid(s) or vector(s) which carry the chondroitinbiosynthesis genes and can thus potentially provide more stableexpression strains or expression strains that are stable in the absenceof any selective pressure. Accordingly, the present invention includesbacterial strains that comprise one or more copies of any one or more ofthe genes contained within the constructs of the present inventionintegrated into their chromosome.

By way of example, the present inventors have created E. coli K-12 andXcc strains that include one or more of the synthetic genes for thebiosynthesis of chondroitin integrated into their chromosomes. Thepresent invention also includes strains that were created by introducingthe constructs comprising expression vectors of the present inventioninto the strains of the present invention that comprise one or morecopies of the constructs integrated into their chromosome.

In some embodiments, the K4 gene cluster, one or more regions of thecluster, one or more subsets of genes of the cluster, or one or moregenes of the cluster are integrated into the chromosome of anon-pathogenic bacterial host cell as described herein using a constructof the invention and methods described herein. In some embodiments, twoor more copies of the gene cluster, region, subset, or gene areintegrated into the chromosome of a non-pathogenic bacterial host cell.In some embodiments, 2 to 20; 2 to 19; 2 to 18; 2 to 17; 2 to 16; 2 to15; 2 to 14; 2 to 13; 2 to 12; 2 to 11; 2 to 10; 2 to 9; 2 to 8; 2 to 7;2 to 6; 2 to 5; 2 to 4; or 2 to 3 copies of the gene cluster, region,subset, or gene are integrated into the chromosome of a non-pathogenicbacterial host cell. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 copies of the gene cluster,region, subset, or gene are integrated into the chromosome of anon-pathogenic bacterial host cell. In some embodiments, the two or morecopies are integrated into the host chromosome using the same construct.In some embodiments, the two or more copies are integrated into the hostchromosome using different constructs. In some embodiments, a promoteris also integrated into the host chromosome to control expression of thegene cluster, region, subset, or gene that is integrated into the hostchromosome. In some embodiments, the two or more copies that areintegrated into the host chromosome are expressed from the same promoteror from different promoters. In some embodiments, two or more copies ofa region 2 gene selected from the group consisting of kfoA, kfoB, kfoC,kfoF, kfoG, and a combination thereof are integrated into a hostchromosome. In some embodiments, two or more copies of kfoA, kfoB, kfoC,kfoF, and kfoG are integrated into a host chromosome. In someembodiments, two or more copies of region 1, region 3, or one or moregenes from region 1 or region 3 are integrated into a host chromosome.In some embodiments, genes contained within a construct of the inventionare integrated into the chromosome of a bacterial host cell that alsocontains one or more constructs of the invention comprising genes thatare not integrated into the chromosome.

Examples of such strains are described in detail in Examples 10-13 and20-21. The constructs and strains described can be used for productionof chondroitin.

Examples of the strains of the present invention include, withoutlimitation, E. coli K-12 strains MSC279, MSC280, MSC322, MSC323, MSC324,MSC325, MSC328, MSC346, MSC356, MSC359, MSC392, MSC402, MSC403, MSC404,MSC405, MSC410, MSC411, MSC436, MSC437, MSC438. MSC439. MSC458. MSC459,MSC460, MSC466, MSC467, MSC498, MSC499, MSC500, MSC510, MSC511, MSC522,MSC526, MSC537, MSC551, MSC561, MSC562, MSC563, MSC564, MSC566, MSC567,MSC619, MSC625, MSC627, MSC640, MSC641, MSC643, MSC646, MSC650, MSC656,MSC657, MSC658, MSC659, MSC660, MSC669, MSC670, MSC671, MSC672, MSC673,MSC674, MSC675, MSC676, MSC677, MSC678, MSC679, MSC680, MSC681, MSC682,MSC683, MSC684, MSC687, MSC688, MSC689, MSC690, MSC691, MSC692, MSC693,MSC694, MSC700, MSC701, MSC702, MSC722, MSC723, and MSC724; E. coli Bstrains MSC315, MSC316, MSC317, MSC319, and MSC347; and X. campestrisstrains MSC326, MSC348, MSC350, MSC480, MSC461, MSC469 and MSC494.

The present invention is directed to a method for producing achondroitin, comprising transferring any one or more of the constructsof the present invention to a non-pathogenic bacterial host cell, andculturing the bacterial host cell under fermentation conditions whereinthe chondroitin is produced by the bacterial host cell.

The present invention is directed to a method for producing achondroitin, comprising culturing a non-pathogenic host cell comprisingany one or more of the constructs of the present invention underfermentation conditions sufficient for production of chondroitin.

The present invention includes a method for producing an unsulfatedchondroitin. The method comprises culturing a non-pathogenic bacterialhost cell of the invention under fermentation conditions sufficient forproduction of unsulfated chondroitin. In some embodiments, the methodcomprises transferring the constructs of the present invention to anon-pathogenic bacterial host cell and culturing the bacterial host cellunder fermentation conditions, which results in the production ofunsulfated chondroitin by the bacterial host cell.

Various embodiments are described in examples 7-15. Specifically,Examples 6-9, 11, 13 and 14 present data demonstrating production ofchondroitin when the constructs of the present invention are transformedinto host cells and Examples 10-15 present data demonstrating productionof chondroitin when the constructs of the present invention areintegrated into the chromosome of the host cell.

Depending on the specific construct and the combination of genestherein, it is possible to produce chondroitin that is fructosylated ornon-fructosylated (see Examples 6 and 7). Furthermore, depending on thespecific construct and the combination of genes therein, the recombinantchondroitin can be secreted into the culture medium or retained in anintracellular location (See Example 9).

Methods of culturing bacterial cells and compositions of culture mediaare well known in the art and can be used in the present invention. Foroptimal production of recombinant chondroitin, various cultureparameters such as temperature, pH, dissolved oxygen concentration,inducer concentration and duration of culture post-induction, as well ascomposition of the media including content of nutrients and saltstherein should be optimized. Example 8 describes the recombinantproduction of chondroitin in a variety of growth media, temperatures andinduction conditions. Based on this information, further optimization ofsuch parameters will be readily apparent to one skilled in the art. Insome embodiments, the bacterial host cell is cultured at 20° C. to 37°C., e.g., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27°C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36°C. or 37° C. In some embodiments, the culture medium comprises yeastextract, a protein digest, a potassium phosphate, and water. In someembodiments, the culture medium comprises glycerine (also known asglycerol). In some embodiments, 1 g/L to 50 g/L, 5 g/L to 50 g/L or 15g/L to 50 g/L of the unsulfated chondroitin is secreted from thebacterial host cell in 24 to 72 hours.

In some embodiments, a method for producing a chondroitin of theinvention further comprises recovering the chondroitin from thebacterial host cell.

In some embodiments, a method for producing a chondroitin of theinvention further comprises recovering the chondroitin fromextracellular culture medium. Chondroitin can be recovered fromfermentation broth by alcohol precipitation or any technique known inthe art, including without limitation, lyophilization to obtain drypowder.

In some embodiments, a method for producing a chondroitin can includethe step of purifying the recovered chondroitin. Purification ofchondroitin can be accomplished by any technique known in the art,including, for example, alkali treatment, acid treatment, proteinasestreatment, chromatography, extraction, solvent extraction, membraneseparation, electrodialysis, reverse osmosis, distillation,precipitation, chemical derivatization, crystallization,ultrafiltration, and/or precipitation of the polysaccharide usingorganic solvents. See, for example. Taniguchi, N., 1982. Isolation andanalysis of glycosaminoglycans. Pages 20-40 in: Glycosaminoglycans andProteoglycans in Physiological and Pathological Process of Body Systems.R. S. Varma and R. Varma, ed. Karger, Basel, Switzerland; Fraquharson etal., Oral. Microbiol. Immunol. 2000; 15:151-157; Manzoni et al., J.Bioact. Comp. Polm. 1993; 8:251-257; Manzoni et al., Biotechnol. Letters2000; 22:759-766; Johns et al., Aust. J. Biotechnol. 1991; 5:73-77; eachof these documents is incorporated by reference herein in its entirety.Examples of precipitation solvents can include, without limitation,acetone, methanol, ethanol, or isopropanol.

In some embodiments, the method for producing a chondroitin furthercomprises sulfating the chondroitin.

The present invention is directed to a method for producing achondroitin sulfate, comprising producing a chondroitin by a method ofthe present invention and sulfating the chondroitin.

Sulfation can be done chemically or enzymatically. Several proceduresare known in the art for chemical sulfation of polysaccharides, any oneof which can be used here. For example, sulfation can be accomplished bysolubilization of the polysaccharide into an organic solvent followed byreacting with a sulfating agent under a controlled temperature. Examplesof solubilization solvents can include, without limitation, formamide,N,N-dimethylformamide (DMF), pyridine, or dimethylsulfoxide. Examples ofsulfating agents can include, without limitation, chlorosulfonic acid,sulfur trioxide, and various sulfur trioxide-amine complexes. Examplesof amines suitable for the sulfur trioxide-amine complexes include,without limitation, pyridine, DMF, trimethylamine, triethylamine (TEA)and piperidine. In some embodiments, upon sulfation of recombinantchondroitin, the sulfated product contains 5.0 to 7.5% of sulfur contentwhich corresponds to that of natural chondroitin sulfate. In furtherembodiments, the sulfated product does not undergo significantdepolymerization. Example 15 describes a method for chemical sulfationof recombinant chondroitin. In some embodiments, sulfating a chondroitinproduced by a method of the invention comprises mixingsulfurtrioxide-triethylamine complex or chlorosulfonic acid with thechondroitin in formamide.

The present invention is directed to a recombinant chondroitin or arecombinant chondroitin sulfate produced by any of the methods describedherein.

The present invention is directed to a composition comprising arecombinant chondroitin or a recombinant chondroitin sulfate produced byany of the methods described herein.

In some embodiments, the composition can include a supplement such asglucosamine, glucosamine sulfate or glucosamine hydrochloride.Glucosamine (2-acetamido-2-deoxyglucose) is a naturally occurringcompound that is found in cartilage. Glucosamine sulfate is a normalconstituent of glycosaminoglycans in the cartilage matrix and synovialfluid. Some clinical trials support the use of glucosamine sulfate inthe treatment of osteoarthritis, particularly that of the knee(Herrero-Beaumont et al. Arthritis Rheum. 2007; 56:555-67; Bruyere etal., Osteoarthritis Cartilage 2008; 16:254-60). It has been proposedthat the sulfate moiety provides clinical benefit in the synovial fluidby strengthening cartilage and aiding glycosaminoglycan synthesis(Silbert Glycobiology 2009; 19:564-567). Glucosamine is commonlyprovided in combination with chondroitin sulfate in nutritionalsupplements intended to promote joint health and as a treatment forosteoarthritis.

In some embodiments, the present invention includes methods ofmaintaining healthy joint function in a subject. In other embodiments,the present invention includes methods for treating or preventingosteoarthritis, interstitial cystitis and/or synovitis. These methodscomprise administering to the subject the compositions comprising therecombinant chondroitin sulfate described above. The compositions of thepresent invention can be generally administered in therapeuticallyeffective amounts.

The present invention is directed to an antibody or antibody fragmentthat selectively binds to a protein encoded by a gene of the K4chondroitin biosynthetic gene cluster. These antibodies and antibodyfragments can be used to confirm the expression of genes of the K4chondroitin biosynthetic gene cluster in the bacterial hosts. In someembodiments, the antibody or antibody fragment binds to an amino acidsequence selected from the group consisting of SEQ ID NO:92 of KpsF, SEQID NO:93 of KpsE, SEQ ID NO:94 of KpsD, SEQ ID NO:95 of KpsU, SEQ IDNO:96 of KpsC, SEQ ID NO:97 of KpsS, SEQ ID NO:91 of KpsT, SEQ ID NO:83of KfoA, SEQ ID NO:84 of KfoB, SEQ ID NO:85 of KfoC, SEQ ID NO:86 ofKfoI (Orf3), SEQ ID NO:87 of KfoE, SEQ ID NO:88 of KfoH (Orf1), SEQ IDNO:89 of KfoF, and SEQ ID NO:90 of KfoG. The generation of theantibodies is described in detail in Example 5.

Fermentation Media and Conditions

In the method for production of chondroitin, a microorganism having agenetic modification described herein is cultured in a fermentationmedium to produce chondroitin. An appropriate, or effective,fermentation medium refers to any medium in which a genetically modifiedmicroorganism of the present invention, when cultured, is capable ofproducing chondroitin. Such a medium is typically an aqueous mediumcomprising assimilable carbon, nitrogen and phosphate sources. Such amedium can also include appropriate salts, minerals, metals and othernutrients. Exemplary media are described below and in the Examplessection. It should be recognized, however, that a variety offermentation conditions are suitable and can be selected by thoseskilled in the art.

Sources of assimilable carbon which can be used in a suitablefermentation medium include, but are not limited to, sugars and theirpolymers, including dextrin, sucrose, maltose, lactose, glucose,fructose, mannose, sorbose, arabinose and xylose; fatty acids; organicacids such as acetate; primary alcohols such as ethanol and n-propanol;and polyalcohols such as glycerine. Carbon sources in the presentinvention include polyalcohols, monosaccharides, disaccharides, andtrisaccharides. In some embodiments, the carbon source is glycerine.

The concentration of a carbon source, such as glycerine, in thefermentation medium should promote cell growth, but not be so high as torepress growth of the microorganism used. Typically, fermentations arerun with a carbon source, such as glycerine, being added at levels toachieve the desired level of growth and biomass, but maintained at lowconcentration levels (below 1 g/L) to avoid accumulation of organicacids, specifically acetate. In other embodiments, the concentration ofa carbon source, such as glycerine, in the fermentation medium isgreater than 1 g/L, greater than 2 g/L, or greater than 5 g/L. Inaddition, the concentration of a carbon source, such as glycerine, inthe fermentation medium is typically less than 100 g/L, less than 50g/L, or less than 20 g/L. It should be noted that references tofermentation component concentrations can refer to both initial and/orongoing component concentrations. In some cases, it can be desirable toallow the fermentation medium to become depleted of a carbon sourceduring fermentation.

Sources of assimilable nitrogen which can be used in a suitablefermentation medium include, but are not limited to, simple nitrogensources, organic nitrogen sources and complex nitrogen sources. Suchnitrogen sources include anhydrous ammonia, ammonium salts andsubstances of animal, vegetable and/or microbial origin. Suitablenitrogen sources include, but are not limited to, protein hydrolysates,microbial biomass hydrolysates, peptone, yeast extract, ammoniumsulfate, ammonium hydroxide, urea, and amino acids. Typically, theconcentration of the nitrogen sources in the fermentation medium isgreater than 0.1 g/L, greater than 0.25 g/L, or greater than 1.0 g/L.Beyond certain concentrations, however, the addition of a nitrogensource to the fermentation medium is not advantageous for the growth ofthe microorganisms. As a result, the concentration of the nitrogensources in the fermentation medium is less than 20 g/L, less than 10g/L, or less than 5 g/L. Further, in some instances it can be desirableto allow the fermentation medium to become depleted of the nitrogensources during fermentation.

The effective fermentation medium can contain other compounds such asdefoamers, inorganic salts, vitamins, trace metals and/or growthpromoters. Such other compounds can also be present in carbon, nitrogenor mineral sources in the effective medium or can be added specificallyto the medium.

The fermentation medium can also contain a suitable phosphate source.Such phosphate sources include both inorganic and organic phosphatesources. Phosphate sources include, but are not limited to, phosphatesalts such as mono or dibasic sodium and potassium phosphates, ammoniumphosphate and mixtures thereof. Typically, the concentration ofphosphate in the fermentation medium is greater than 1.0 g/L, greaterthan 2.0 g/L, or greater than 5.0 g/L. Beyond certain concentrations,however, the addition of phosphate to the fermentation medium is notadvantageous for the growth of the microorganisms. Accordingly, theconcentration of phosphate in the fermentation medium is typically lessthan 20 g/L, less than 15 g/L, or less than 10 g/L.

A suitable fermentation medium can also include a source of magnesium.In some embodiments, the source of magnesium is in the form of aphysiologically acceptable salt, such as magnesium sulfate heptahydrate,although other magnesium sources in concentrations which contributesimilar amounts of magnesium can be used. Typically, the concentrationof magnesium in the fermentation medium is greater than 0.5 g/L, greaterthan 1.0 g/L, or greater than 2.0 g/L. Beyond certain concentrations,however, the addition of magnesium to the fermentation medium is notadvantageous for the growth of the microorganisms. Accordingly, theconcentration of magnesium in the fermentation medium is typically lessthan 10 g/L, less than 5 g/L, or less than 3 g/L. Further, in someinstances it can be desirable to allow the fermentation medium to becomedepleted of a magnesium source during fermentation.

The fermentation medium can also include a biologically acceptablechelating agent, such as the dihydrate of trisodium citrate or citricacid. In such instance, the concentration of a chelating agent in thefermentation medium is greater than 0.1 g/L, greater than 0.2 g/L,greater than 0.5 g/L, or greater than 1 g/L. Beyond certainconcentrations, however, the addition of a chelating agent to thefermentation medium is not advantageous for the growth of themicroorganisms. Accordingly, the concentration of a chelating agent inthe fermentation medium is typically less than 10 g/L, less than 5 g/L,or less than 2 g/L.

The fermentation medium can also initially include a biologicallyacceptable acid or base to maintain the desired pH of the fermentationmedium. Biologically acceptable acids include, but are not limited to,hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid andmixtures thereof. Biologically acceptable bases include, but are notlimited to, anhydrous ammonia, ammonium hydroxide, sodium hydroxide,potassium hydroxide and mixtures thereof. In some embodiments of thepresent invention, the base used is ammonium hydroxide.

The fermentation medium can also include a biologically acceptablecalcium source, including, but not limited to, calcium chloride.Typically, the concentration of the calcium source, such as calciumchloride, dihydrate, in the fermentation medium is within the range offrom 5 mg/L to 2000 mg/L, 20 mg/L to 1000 mg/L, or 50 mg/L to 500 mg/L.

The fermentation medium can also include sodium chloride. Typically, theconcentration of sodium chloride in the fermentation medium is withinthe range of from 0.1 g/L to 5 g/L, 1 g/L to 4 g/L, or 2 g/L to 4 g/L.

As previously discussed, the fermentation medium can also include tracemetals. Such trace metals can be added to the fermentation medium as astock solution that, for convenience, can be prepared separately fromthe rest of the fermentation medium. A suitable trace metals stocksolution for use in the fermentation medium is shown below in Tables 1Aand 1B. Typically, the amount of such a trace metals solution added tothe fermentation medium is greater than 1 mL/L, greater than 5 mL/L, orgreater than 10 mL/L. Beyond certain concentrations, however, theaddition of a trace metals to the fermentation medium is notadvantageous for the growth of the microorganisms. Accordingly, theamount of such a trace metals solution added to the fermentation mediumis typically less than 100 mL/L, less than 50 mL/L, or less than 30mL/L. It should be noted that, in addition to adding trace metals in astock solution, the individual components can be added separately, eachwithin ranges corresponding independently to the amounts of thecomponents dictated by the above ranges of the trace metals solution.

As shown below in Table 1A, a suitable trace metals solution for use inthe present invention can include, but is not limited to ferroussulfate, heptahydrate; cupric sulfate, pentahydrate; zinc sulfate,heptahydrate; sodium molybdate, dihydrate; cobaltous chloride,hexahydrate; and manganous sulfate, monohydrate. Hydrochloric acid isadded to the stock solution to keep the trace metal salts in solution.

TABLE 1A TRACE METALS STOCK SOLUTION A CONCENTRATION COMPOUND (mg/L)Ferrous sulfate, heptahydrate 280 Cupric sulfate, pentahydrate 80 Zinc(II) sulfate, heptahydrate 290 Sodium molybdate 240 Cobaltous chloride,hexahydrate 240 Manganous sulfate, monohydrate 170 Hydrochloric acid 0.1ml

Another suitable trace metal solution for use in the present inventionis shown in Table 1B and can include, but is not limited to, ferricchloride, hexahydrate; zinc chloride; cobalt chloride, hexahydrate;sodium molybdate; manganese chloride; boric acid; and citric acid as achelating agent.

TABLE 1B TRACE METALS STOCK SOLUTION B CONCENTRATION COMPOUND (g/L)Ferric Chloride, hexahydrate 27 Zinc Chloride 1.3 Cobalt Chloride,hexahydrate 2 Sodium molybdate 2 Manganese Chloride 3.3 Boric acid 0.5Citric acid 33

The fermentation medium can also include vitamins. Such vitamins can beadded to the fermentation medium as a stock solution that, forconvenience, can be prepared separately from the rest of thefermentation medium. A suitable vitamin stock solution for use in thefermentation medium is shown below in Table 2. Typically, the amount ofsuch vitamin solution added to the fermentation medium is greater than 1ml/L, greater than 5 mL, or greater than 10 ml/L. Beyond certainconcentrations, however, the addition of vitamins to the fermentationmedium is not advantageous for the growth of the microorganisms.Accordingly, the amount of such a vitamin solution added to thefermentation medium is typically less than 50 ml/L, less than 30 ml/L,or less than 20 ml/L. It should be noted that, in addition to addingvitamins in a stock solution, the individual components can be addedseparately, each within the ranges corresponding independently to theamounts of the components dictated by the above ranges of the vitaminstock solution.

As shown in Table 2, a suitable vitamin solution for use in the presentinvention can include, but is not limited to, biotin, calciumpantothenate, inositol, pyridoxine-HCl and thiamine-HCl.

TABLE 2 VITAMIN STOCK SOLUTION CONCENTRATION COMPOUND (mg/L) Biotin 10Calcium pantothenate 120 Inositol 600 Pyridoxine-HCl 120 Thiamine-HCl120

Microorganisms of the present invention can be cultured in conventionalfermentation modes, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous. In some embodiments, thefermentation is carried out in fed-batch mode. In such a case, duringfermentation some of the components of the medium are depleted. It ispossible to initiate the fermentation with relatively highconcentrations of such components so that growth is supported for aperiod of time before additions are required. The preferred ranges ofthese components are maintained throughout the fermentation by makingadditions as levels are depleted by fermentation. Levels of componentsin the fermentation medium can be monitored by, for example, samplingthe fermentation medium periodically and assaying for concentrations.Alternatively, once a standard fermentation procedure is developed,additions can be made at timed intervals corresponding to known levelsat particular times throughout the fermentation. As will be recognizedby those in the art, the rate of consumption of nutrient increasesduring fermentation, as the cell density of the medium increases.Moreover, to avoid introduction of foreign microorganisms into thefermentation medium, addition is performed using aseptic additionmethods, as are known in the art. In addition, a small amount ofanti-foaming agent can be added during the fermentation.

The temperature of the fermentation medium can be any temperaturesuitable for growth and production of chondroitin. For example, prior toinoculation of the fermentation medium with an inoculum, thefermentation medium can be brought to and maintained at a temperature inthe range of from 20° C. to 45° C., 25° C. to 40° C., or 28° C. to 32°C.

The pH of the fermentation medium can be controlled by the addition ofacid or base to the fermentation medium. In such cases when ammonia isused to control pH, it also conveniently serves as a nitrogen source inthe fermentation medium. In some embodiments, the pH is maintained from3.0 to 8.0, 5.5 to 7.5, or 6.0 to 7.

The fermentation medium can also be maintained to have a constantdissolved oxygen content during the course of fermentation to maintaincell growth and to maintain cell metabolism for production ofchondroitin. The oxygen concentration of the fermentation medium can bemonitored using known methods, such as through the use of an oxygenelectrode. Oxygen can be added to the fermentation medium using methodsknown in the art, for example, through agitation and aeration of themedium by stirring, shaking or sparging. In some embodiments, the oxygenconcentration in the fermentation medium is in the range of from 10% to200% of the saturation value of oxygen in the medium based upon thesolubility of oxygen in the fermentation medium at atmospheric pressureand at a temperature in the range of from 20° C. to 40° C. Periodicdrops in the oxygen concentration below this range can occur duringfermentation, however, without adversely affecting the fermentation.

Although aeration of the medium has been described herein in relation tothe use of air, other sources of oxygen can be used. Particularly usefulis the use of an aerating gas which contains a volume fraction of oxygengreater than the volume fraction of oxygen in ambient air. In addition,such aerating gases can include other gases which do not negativelyaffect the fermentation.

In an embodiment of the fermentation process of the present invention, afermentation medium is prepared as described above. This fermentationmedium is inoculated with an actively growing culture of geneticallymodified microorganisms of the present invention in an amount sufficientto produce, after a reasonable growth period, a high cell density.Typical inoculation cell densities are within the range of from 0.001g/L to 10 g/L, 0.01 g/L to 5 g/L, or 0.05 g/L to 1.0 g/L, based on thedry weight of the cells. In production scale fermentors, however,greater inoculum cell densities are preferred. The cells are then grownto a cell density in the range of from 10 g/L to 150 g/L, 20 g/L to 80g/L, or 50 giL to 70 g/L. The residence times for the microorganisms toreach the desired cell densities during fermentation are typically lessthan 200 hours, less than 120 hours, or less than 96 hours.

In one mode of operation of the present invention, the carbon sourceconcentration, such as the glycerine concentration, of the fermentationmedium is monitored during fermentation. Glycerine concentration of thefermentation medium can be monitored using known techniques, such as,for example, use of the high pressure liquid chromatography, which canbe used to monitor glycerine concentration in the supernatant, e.g., acell-free component of the fermentation medium. As stated previously,the carbon source concentration should be kept below the level at whichcell growth inhibition occurs. Although such concentration can vary fromorganism to organism, for glycerine as a carbon source, cell growthinhibition occurs at glycerine concentrations greater than at about 60g/L, and can be determined readily by trial. Accordingly, when glycerineis used as a carbon source the glycerine is preferably fed to thefermentor and maintained below detection limits. Alternatively, theglycerine concentration in the fermentation medium is maintained in therange of from 1 g/L to 100 g/L, 2 g/L to 50 g/L, or 5 g/L to 20 g/L.Although the carbon source concentration can be maintained withindesired levels by addition of, for example, a substantially pureglycerine solution, it is acceptable to maintain the carbon sourceconcentration of the fermentation medium by addition of aliquots of theoriginal fermentation medium. The use of aliquots of the originalfermentation medium can be desirable because the concentrations of othernutrients in the medium (e.g. the nitrogen and phosphate sources) can bemaintained simultaneously. Likewise, the trace metals concentrations canbe maintained in the fermentation medium by addition of aliquots of thetrace metals solution.

Chondroitin Recovery

Once chondroitin is produced by a fermentation methodology, it can berecovered for subsequent use. The present inventors have shown thatchondroitin can be present in a cell-free form in culture media(“secreted chondroitin”) and/or associated with the cells. Chondroitinthat is associated with the cells can be associated with the cellsurface (“cell-surface chondroitin”) and/or retained within the cell(“intracellular chondroitin”).

With respect to “secreted chondroitin,” the recovery of chondroitin canbe accomplished by cell removal followed by alcohol precipitation of thecell-free culture media, or any technique known in the art, includingwithout limitation, lyophilization of the cell-free culture media toobtain dry powder.

With respect to “cell-surface chondroitin,” the recovery of chondroitincan additionally include a step of detaching the chondroitin from thecell surface followed by a cell removal step that removes the cells fromthe culture media that contains the free chondroitin. With respect to“intracellular chondroitin,” the recovery can additionally include astep of permeabilizing or lysing the cells, followed by a step thataccomplishes removal of the lysed or permeabilized cells from theculture media that now contains the liberated chondroitin. Chondroitincan be recovered from the culture media by alcohol precipitation of theculture media, or any technique known in the art, including withoutlimitation, lyophilization of the culture media to obtain dry powder.

Additionally, the recovered chondroitin polymer can be depolymerized toreduce the molecular weight of the polymer. Depolymerization of achondroitin can be accomplished by any technique known in the art,including without limitation, acidic depolymerization. See, for example,Tommeraas and Melander, Biomacromolecules 2008; 9:1535-1540. A recoveredchondroitin can be depolymerized, for example, to produce a chondroitinwith a similar or identical molecular weight to an animal-derivedchondroitin and/or to aid in sulfation of a recovered non-sulfatedchondroitin. For example, a recovered chondroitin can be depolymerizedto obtain a polymer with a molecular weight of 5 kDa to 100 kDa,preferably, 10 kDa to 70 kDa, more preferably, 20 kDa to 40 kDa.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

As used herein the term “gene” refers to a nucleic acid fragment (orpolynucleotide) that is capable of being expressed as a specificprotein, optionally including regulatory sequences preceding (5′non-coding sequences) and following (3′ noncoding sequences) the codingsequence. “Native gene” refers to a gene that is found in nature withits own regulatory sequences. “Endogenous gene” refers to a native genein its natural location in the genome of an organism.

As used herein the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence.

“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coxiing sequences), within, or downstream (3′non-coding sequences) of a coding sequence, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences can include promoters,translation leader sequences, introns, polyadenylation recognitionsequences, RNA processing sites, effector binding sites and stem-loopstructures.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters can be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters can direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths can have identical promoter activity.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression can also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “construct,” “plasmid”, “vector” and “cassette” refer to anextra chromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circular, orlinear, double stranded DNA fragments. Such elements can be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutvariation in the amino acid sequence of an encoded polypeptide. Theskilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in usage of nucleotide codons to specify a givenamino acid. Therefore, when synthesizing a gene for improved expressionin a host cell, it is desirable to design the gene such that itsfrequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic add fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel. F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987). Each of these documentsis incorporated by reference herein in there entirety.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1 Genetics of K4 Capsule Biosynthesis

The E. coli K4 capsule is categorized as a “group 2” capsule. Asreviewed by Whitfield (Annu Rev Biochem. 2006; 75:39-68), the synthesisof E. coli group 2 capsules is directed by a set of proteins encoded bygene clusters having a common genetic organization consisting of threeregions. The predicted structure (prior to the current invention) of theE. coli K4 capsule gene cluster is shown in the FIG. 2. Region 1 wasexpected to contain six genes, kpsFEDUCS, and region 3 was expected tocontain two genes, kpsM and kpsT. Based on sequence homology with knownproteins, kpsF and kpsU genes were predicted to encode proteins thatcatalyze steps in the biosynthesis of the sugar nucleotide CMP-Kdo. Arole of CMP-Kdo biosynthesis of group 2 capsules in E. coli has beenproposed (Roberts, Annu. Rev. Microbiol. 1996: 50:285-315) but has notbeen demonstrated experimentally (Whitfield, Annu Rev Biochem. 2006;75:39-68). The kpsM, kpsT, kpsD, kpsE, kpsC and kpsS genes were positedto encode proteins required for translocation of the capsularpolysaccharide from the cell cytoplasm, where polymerization of sugarprecursors occurs, to the cell surface where the mature capsularpolysaccharide is believed to be anchored to the outer cell membranethrough a covalent linkage to a lipid component of that membrane(Roberts, Annu. Rev. Microhiol. 1996; 50:285-315; Whitfield, Annu RevBiochem. 2006; 75:39-68). For the E. coli K4 capsule, as for most E.coli group 2 capsules, the structure of the covalent linkage between thepolysaccharide and the lipid component of the capsule has not beendetermined experimentally. Moreover, the identity of the lipid componentis not known. The region 1 and region 3 genes, and the proteins thatthey encode, are highly conserved among E. coli strains that producecapsules having very diverse polysaccharide compositions and structures(Whitfield, Annu Rev Biochem. 2006; 75:39-68). The genes contained inregion 2 of group 2 capsule clusters in E. coli include genes thatencode enzymes for sugar nucleotide precursor biosynthesis and forpolymerization of these precursors, and region 2 thereby determines thestructure of the capsular polysaccharide. Other genes in region 2 ofgroup 2 capsule clusters in E. coli encode proteins for which functionsare unknown and have not been demonstrated to have a role in capsulebiosynthesis. The sequence of region 2 of the E. coli K4 capsule genecluster as described by Ninomiya et al. (J. Biol. Chem. 2002;277:21567-21575, GenBank AB079602) contains 7 annotated open readingframes (kfoABCDEFG) predicted to encode proteins. An insertion element,IS2, is located between genes kfoC and kfoD.

As a preliminary step in design of synthetic coding sequences for the K4capsule biosynthetic genes, the intergenic sequences separating eachgene pair were examined. Based on this sequence analysis, it appearedlikely that there were, within this region, at least two additionalopen-reading-frames (ORFs) encoding proteins that are likely to beexpressed and potentially related to capsule biosynthesis. Based on theNinomiya et al. sequence, the following intergenic distances wereobtained: kfoA-kfoB: 186 bp; kfoB-kfoC: 297 bp; kfoC-IS2: 29 bp;IS2-kfoD: 9 bp; kfoD-kfoE: 389 bp; kfoE-kfoF: 818 bp; kfoF-kfoG: 431 bp.One open reading frame was identified in each of the three largestintergenic regions.

Most of the kfoD-kfoE region is encompassed by a 390 bp ORF, termed“ORF3”, which initiates 10 nucleotides after the stop codon of kfoD andterminates within the coding region of kfoE. That is, the putative orf3gene overlaps the kfoE gene by 10 nucleotides. This ORF is initiated byan ATG and a second possible ATG start is located, in frame, 9 bpdownstream. Both of these possible starts lack a recognizableShine-Dalgarno (SD) sequence (Shine and Dalgarno Proc. Natl. Acad. Sci.USA. 1974; 71:1342-6). When the protein product of orf3 was used in aBLAST search, 8 “goodx” hits, i.e. Scores >138, E values <3e-31 wereobtained. Two of these hits were to proteins encoded by Pasteurellamultocida genes (bcbF & ecbF) located in gene clusters for capsulebiosynthesis. These P. multocida capsule gene clusters are diagrammed inFIG. 3A along with the K4 region 2 genes according to the sequence ofNinomiya et al. as analyzed by the present inventors. Alignment ofprotein sequences for Orf3, BcbF and EcbF proteins is shown in FIG. 3B.These P. multocida sequences come from a serotype B strain (M1404) and aserotype E strain (P1234), respectively. Composition of the semtype Ecapsule is unknown while the serotype B capsule is reported to becomposed of mannose, galactose and arabinose but no structure has beenreported (Townsend et al., J. Clin. Microbiol. 2001:39:924-929).

In the kfoE-kfoF region, a 630 bp ORF termed “ORF1” is present as shownin FIG. 3A. The ATG start codon for this ORF overlaps with the TGA stopcodon of the upstream kfoE gene. Seven base pairs upstream of this ATG,within the coding sequence for KfoE, a strong SD sequence (AGGAGG) ispresent. Thus, the circumstantial evidence is strong that this ORFshould be expressed. BLAST results for the protein encoded by ORF1include strong hits to P. multocida genes (bcbE & ecbE) that areadjacent to the P. multocida gene hits obtained with ORF3. That is, bothputative ORFs 1 and 3 of the K4 cluster have homologs within two P.multocida gene clusters encoding capsular polysaccharides. Alignment ofORF1, BcbE and EcbE protein sequences is shown in FIG. 3B.

In the kfoF-kfoG region, a 384 bp ORF, termed “ORF2” is present. The ATGstart codon of this ORF is preceded by a GG sequence 15 bp upstream,which could provide a weak SD sequence. No significant hits were foundin BLAST searches with this protein sequence. This suggested that thisORF may not encode a polypeptide that is actually produced.

It is interesting to note that two other K4 capsule cluster genes fromregion 2 (kfoD and kfoE) have homology to P. multocida genes located inthe P. multocida P1234 and P. multocida M1404 capsule gene clusters. Theprotein encoded by kfoD shares homology with EcbD and BcbD and similarlythe kfoE gene product shares homology with EcbG and BcbG. Thus, as shownin FIG. 3A, the four contiguous K4 genes (kfoD. orf3, kfoE, and orf1)have homologs among the contiguous P. multocida serotype B genes bcbDEFGand the P. multocida serotype E genes ecbDEFG. As noted above, these twoPasteurella strains are not chondroitin producers and the role, if any,which the K4 genes kfoD, orf3, kfoE, and orf1 play in chondroitinbiosynthesis was unknown prior to this invention.

The fact that the kfoD, orf3, kfoE, orf1 gene set in K4 is immediately(9 bp) preceded by IS2 raises numerous possibilities regarding theirorigin and role in chondroitin capsule synthesis. Without wishing to bebound by theory, the present inventors contemplate that the K4 region 2gene cluster has arisen via IS2-mediated recombination/insertion into aparental, chondroitin-producing, cluster comprised of kfoABCFG. Further,the present inventors hypothesized that the kfoD, orf3, kfoE and orf1genes might be involved in the fructosylation of the chondroitinbackbone. The fructosylation is the one obvious structural differencebetween the P. multocida serotype F capsule and the E. coli K4 capsule.It is possible that the significant difference in genetic structurebetween the two, i.e. the presence or absence of the kfoD, orf3, kfoE,orf1 gene set, is a reflection of that structural difference. By analogyto the capsule biosynthetic gene cluster of the P. multocidachondroitin-producing serotype F strain P4182 (Townsend et al., J. Clin.Microbiol. 2001; 39:924-929), it may be possible that the only relevantK4 region 2 genes for production of the chondroitin backbone might bekfoA, kfoB, kfoC, kfoF and kfoG. As described in Examples 6 and 7, thepresent inventors confirmed that the kfoD, orf3, kfoE and orf1 genes arenot required for production of chondroitin and that one or more of thesegenes is essential for fructosylation of chondroitin.

In order to confirm the Ninomiya et al. sequence prior to designingsynthetic coding sequences for the K4 capsule biosynthetic genes, thepresent inventors sequenced region 2 of the K4 capsule gene cluster fromthe E. coli K4 strain ATCC 23502 obtained from ATCC. Genomic DNA wasprepared from a fresh overnight culture of ATCC strain 23502 using theQiagen Genomic DNA Kit (Qiagen Inc., Valencia, Calif.) according to thevendor protocol. Aliquots of genomic DNA, sheared by passage (fivetimes) through a 20 gauge needle were used as template in PCR reactionsto produce a series of 6 overlapping PCR products ranging in size from2.2 kB to 2.7 kB. The products of the PCR reactions were purified usingthe Qiagen QIAquick PCR Purification Kit according to the vendorprotocol and sent to a commercial vendor (Biotechnology Resource Center,DNA Sequencing Facility, Cornell University, Ithaca, N.Y.) for DNAsequence determination. The sequences of these 6 overlapping PCRproducts spanned the region 2 sequence as determined by Ninomiya et al.(2002). On the whole, there was agreement between the sequencedetermined by the inventors and the sequence reported by Ninomiya et al.with a 99.8% identity. However, there were single base pair differences,including substitutions, deletions and insertions, at 26 positions. Someof these differences resulted in substantial differences in thepredicted amino acid sequences of region 2 proteins encoded by the genecluster. The observed nucleotide sequence differences and resultingeffect on predicted protein sequences are shown in FIGS. 4A and 4B.

In order to determine the correct sequence of the K4 capsulebiosynthetic genes, E. coli serotype K4 strain U1-41 was obtained fromthe Statens Serum Institut (Copenhagen, Denmark). U1-41 is theprogenitor of the ATCC 23502 strain and was used to produce thepolysaccharide preparation used for structural determination of the K4polysaccharide (Rodriguez et al., 1988). The sequence of approximately23 kb of DNA spanning regions 1, 2 and 3 of the K4 capsule gene clusterin E. coli U1-41 was determined. This sequence (SEQ ID NO:117) consistsof 23,230 base pairs spanning a region from 125 bp upstream of the ATGtranslational start codon of the kpsF gene of region 1 to 110 bpupstream of the ATG translational start codon of the kpsM gene of region3.

For sequence determination, genomic DNA was prepared from a freshovernight culture of E. coli U1-41 using the Qiagen Genomic DNA Kit(Qiagen Inc., Valencia, Calif.) according to the vendor protocol.Aliquots of genomic DNA, sheared by passage (five times) through a 20gauge needle was used as template in PCR reactions to produce a seriesof 11 overlapping PCR products ranging in size from 2.1 kB to 2.8 kB.These PCR reactions, termed here Reactions 1 through 11, employed thefollowing oligonucleotide primers: Reaction 1; (DHD89 and DHD090),Reaction 2; (DHD091 and DHD092), Reaction 3; (DHD093 and DHD175),Reaction 4; (DHD120 and DHD096), Reaction 5; (DHD097 and DHD098),Reaction 6; (DHD099 and DHD100), Reaction 7; (DHD101 and DHD102),Reaction 8; (DHD103 and DHD104), Reaction 9; (DHD105 and DHD106),Reaction 10; (DHD162 and DHD108), Reaction 11; (DHD169 and DHD110).Sequences of these primers are shown below.

(SEQ ID NO: 118) DHD089 5 > GCACCTCCATGAGACATTGC > 3 (SEQ ID NO: 119)DHD090 5 > CCACTGCCATACGGTTTAGC > 3 (SEQ ID NO: 120) DHD091 5 >GCTTGCCTTTGCAGAAACGG > 3 (SEQ ID NO: 121) DHD092 5 >CCAACAATATCGAGCAGTGG > 3 (SEQ ID NO: 122) DHD093 5 >GTCATTCGTCAGAACGGTGC > 3 (SEQ ID NO: 123) DHD175 5 >CCAGTGCCTGATAATCAGC > 3 (SEQ ID NO: 124) DHD120 5 >GGCTTAACGCTGTGGAAGTC > 3 (SEQ ID NO: 125) DHD096 5 >ATATTGGGATTCCTGGTCGC > 3 (SEQ ID NO: 126) DHD097 5 >ACGACATCAAAGGCTTGACG > 3 (SEQ ID NO: 127) DHD098 5 >ATAGCCCTGAAGCTGAAGCC > 3 (SEQ ID NO: 128) DHD099 5 >CGAGTGATTGCTTGGTATCC > 3 (SEQ ID NO: 129) DHD100 5 >AAACGATTGAGCGGGTTAGC > 3 (SEQ ID NO: 130) DHD101 5 >AGAGTGGTTCAATCCTCTGG > 3 (SEQ ID NO: 131) DHD102 5 >TGTCTTGGCTAATGCTGACG > 3 (SEQ ID NO: 132) DHD103 5 >CGAGTAGTTATCTGGCTCTG > 3 (SEQ ID NO: 133) DHD104 5 >GTCAGTTAGACTCTGATGAC > 3 (SEQ ID NO: 134) DHD105 5 >CTTGAACGGTCCAACTTCAC > 3 (SEQ ID NO: 135) DHD106 5 >AGTTCAGGAGCTTGAATGCG > 3 (SEQ ID NO: 136) DHD162 5 >TTCGCACGCATTTATAGCCG > 3 (SEQ ID NO: 137) DHD108 5 >TCATCTTGCGAGAGCATTCG > 3 (SEQ ID NO: 138) DHD169 5 >CTTCCGCTAAATCCATTACG > 3 (SEQ ID NO: 139) DHD110 5 >AGATCTATTTATCCCTGCGG > 3

PCR Reactions 1, 2, 3, 7, 8, 9, 10 and 11 were performed using PfuUltraII polymerase (STRATAGENE, LaJolla, Calif.) according to the vendorprotocols. In each 100 μL reaction, Pfu reaction buffer (supplied by thevendor) was added to a final concentration of 1×, primers were added toa final concentration of 0.4 μM each, dNTPs were added at a finalconcentration of 250 μM each and 100 ng of U1-41 genomic DNA was addedas template. PCR reactions were performed in a Perkin-Elmer GeneAmp 2400thermocyler using the following cycling parameters: 1 cycle of 2 minutesat 95° C.; 35 cycles of 20 seconds at 95° C., 20 seconds at 55° C., and40 seconds at 72° C.; 1 cycle of 3 minutes at 72° C.; and a hold at 4°C. PCR Reactions 4, 5 and 6 were performed as above with the followingexceptions. In the case of Reactions 5 and 6, the primers were added toa final concentration of 0.5 μM each and the annealing step was at 60°C. instead of 55° C. In the case of Reaction 4, primers were added to afinal concentration of 0.5 μM each and PCR reactions were performed in aRoboCycler® Gradient 96 thermocycler (STRATAGENE, LaJolla, Calif.) usingthe following cycling parameters: 1 cycle of 1 minute at 95° C.; 35cycles of 30 seconds at 95° C., 30 seconds at 52° C., and 1 minute at72° C.; 1 cycle of 5 minutes at 72° C.; and a hold at 6° C.

The products of PCR Reactions 1, 2, 3, 7, 8, 9, 10 and 11 were purifiedusing the Qiagen QIAquick PCR Purification Kit (QIAGEN, Valencia,Calif.) according to the vendor protocol, recovered in 100 μL of EBelution buffer, and then used as templates for sequencing reactions.Products of PCR Reactions 4, 5 and 6 were purified using the QiagenQIAquick PCR Purification Kit (QIAGEN, Valencia, Calif.) according tothe vendor protocol and then further purified by preparative agarose gelelectrophoresis. Fragments were excised from the gels and eluted fromthe gel slices using the QIAquick Gel Extraction Kit (QIAGEN Inc.,Valencia, Calif.) according to the vendor protocol and recovered in 100μL of EB elution buffer. The gel-purified fragments served as templatesfor sequencing reactions. The purified PCR products of reactions 1-11were sent to a commercial vendor (Cornell University Life Sciences CoreLaboratories Center, Cornell University, Ithaca. New York) for DNAsequence determination. The sequences obtained from these 11 overlappingPCR products spanned the region 2 sequence as determined by Ninomiya etal. (2002) and included all of the region 1 and region 3 genes as well.

The sequence of the K4 capsule gene cluster from U1-41 demonstrates thepresence of kpsF, kpsE, kpsD, kpsU, kpsC and kpsS genes in region 1 asis typical for group 2 capsule gene clusters in E. coli. The predictedamino acid sequences of U1-41 KpsF, KpsE, KpsD, KpsU, KpsC and KpsSproteins are homologous to the sequences of these proteins encoded byother E. coli group 2 capsule producers. They all show >95% identity tothe consensus sequences for these proteins and to the E. coli Nissle1917 (serotype K5) KpsF, KpsE, KpsD, KpsU, KpsC and KpsS sequences(Grozdanov et al., J. Bacteriol. 2004; 186:5432-41). The sequence alsoreveals the presence of kpsM and kpsT genes in region 3 as is typicalfor group 2 capsule gene clusters in E. coli. The predicted amino acidsequences of U1-41 KpsM and KpsT proteins are homologous to thesequences of these proteins encoded by other E. coli group 2 capsuleproducers. They all show >90% identity to the consensus sequences forthese proteins and to the E. coli Nissle 1917 (serotype K5) KpsM andKpsT sequences.

The U1-41 DNA sequence includes an approximately 13.5 kb region 2segment that can be aligned to the Ninomiya et al. region 2 sequence andto the sequence determined by the present inventors for region 2 of ATCC23502. The U1-41 sequence and the sequence determined by the presentinventors for region 2 of ATCC 23502 are identical over this span. Nineopen reading frames (ORFs) predicted to be expressed as polypeptides arepresent in the identified region 2, exclusive of the IS2 sequence. Thesenine include the two ORFs, detailed above, that were not previouslyidentified. The genes encoding these ORFs were initially termed hereorf1 and orf3 and are now proposed to be designated as kfoH and kfoI,respectively. FIG. 5 shows the arrangement of the K4 capsule genecluster as determined by the present inventors from the DNA sequences ofATCC 23502 and U1-41. Orf2 mentioned above was not found to exist as aseparate open reading frame in the sequence of region 2 as determined bythe present inventors. In the sequence as determined by the presentinventors, the sequences comprising orf2 are a portion of the codingsequence of kfoG. Frameshifts within the sequence published by Ninomiyaet al. split the kfoG sequence into two smaller open reading frames; thekfoG gene as annotated by Ninomiya et al. and the orf2 sequence asannotated by the present inventors. Thus, orf2 was an artifact of theerroneous sequence published by Ninomiya et al.

Exclusive of the IS2 sequence, the gene cluster contains 17 open readingframes that are predicted to encode proteins. The arrangement of thesegenes is typical for an E. coli group 2 capsule gene cluster (Whitfield2006). Region 1, comprising conserved genes kpsFEDUCS, and region 3,comprising conserved genes kpsMT, flank the 9 open reading frames (andIS2) of region 2. Region 1 and region 3 genes include proteins that arerequired for synthesis and translocation of all group 2 capsules in E.coli. Region 1 also includes two genes (AkF and kpsU) that encodeenzymes that are predicted to catalyze steps in the biosynthesis ofCMP-Kdo. As noted above, a role of CMP-Kdo in biosynthesis of group 2capsules in E. coli has been proposed, but has not been demonstratedexperimentally. In group 2 capsule gene clusters, region 2 genestypically include those that encode serotype-specific proteins thatdetermine the structure of the capsular polysaccharide. Of the ninegenes identified in region 2, three encode proteins with clearly definedactivities relating to capsule biosynthesis: kfoC (chondroitin synthase,i.e., the polymerase), kfoA (UDPGlcNAc epimerase, converts UDPGlcNAc tothe UDPGaNAc precursor) and kfoF (UDPGlc-dehydrogenase, converts UDPGlcto the UDPGlcUA precursor).

Functions remain unknown for other genes present in region 2 of the K4capsule gene cluster: kfoB, kfoG, kfoD. kfoE. kfoH and kfoI. The kfoBencodes a protein that is homologous to proteins encoded by genespresent in capsule clusters of bacteria known to produce otherglycosaminoglycan (GAG) capsules, P. multocida serotypes A, F and D andE. coli serotype K5. Similarly the KfoG protein also has homology toproteins encoded by genes present in the capsule clusters of P.multocida serotypes A, F and D. This circumstantial evidence suggeststhat kfoB and kfoG can play a role in the biosynthesis of theGAG-containing K4 capsule.

In contrast to KfoB and KfoG, prior to this invention, no such evidenceimplicated kfoD, kfoE, kfoH and kfoI as being involved in GAGbiosynthesis. As noted above and described in Examples 6 and 7, thepresent inventors show herein that one or more of these genes (i.e.genes kfoD, kfoE, kfoH and kfoI) is essential for fructosylation of thechondroitin backbone of the K4 capsular polysaccharide, but that none ofthese genes are required for production of the chondroitin backbone.

The insertion element IS2 is present between genes kfoC and kfoD inU1-41 as well as in ATCC 23502. Insertion of IS2 in the observedorientation has been reported to activate expression of downstream genesdue to transcription that originates within IS2 (Glansdorf et al., ColdSpring Harbor Symp. Quant. Biol., 1981; 45:153-156). Thus, withoutwishing to be bound by theory, it is proposed that the presence of IS2could modulate expression of downstream genes kfoD, kfoI, kfoE. kfoH,kfoF and kfoG, but is not predicted to prevent expression of thesegenes.

Example 2 Synthesis of Codon-Optimized E. coli K4 Capsule BiosyntheticGenes

The sequence of the U1-41 K4 capsule gene cluster as determined by thepresent inventors was used as the basis for the design of syntheticgenes to be used for expression in alternative hosts. Syntheticconstructs were designed to allow the expression of one or moresynthetic operons containing the K4 capsule biosynthetic genes and wereoptimized for codon usage based on a consensus preferred codon tablethat employs codons that are acceptable for expression in E. coli, X.campestris, S. elodea and B. subtilis. Table 3A gives codon usage tablesfor E. coli, X. campestris and B. subtilis genomes as well as for E.coli K4 region 2 genes related to K4P capsule biosynthesis, and 53 S.elodea genes including those related to gellan biosynthesis. This tableillustrates the striking use of unfavorable codons in the K4 region 2biosynthetic genes. These codons are not only extremely unfavorable forX. campestris or S. elodea expression but are also unfavorable forexpression in E. coli. For optimal expression in E. coli, it would beexpected that significant codon optimization would be necessary. Basedon comparison of these codon usage tables, a consensus preferred codonusage table was designed, shown in Table 3B for the syntheticchondroitin biosynthetic genes. This codon usage pattern is expected toprovide efficient translation in a wide variety of potential alternativehosts.

TABLE 3A CODON USAGE B. X. S. E. coli E. coli Amino subtilis campestriselodea K-12 K4 acid codon genome genome 53 CDSs genome kfoA-G arg cgg 1615 22 10 5 cga 10 3 4 6 16 cgt 18 15 9 38 22 cgc 21 66 63 40 9 agg 10 22 2 12 aga 26 1 <1 4 36 ser tcg 10 29 39 15 7 tca 24 3 2 12 25 tct 20 32 15 25 tcc 13 18 18 15 8 agt 11 7 5 15 23 agc 23 40 34 28 11 leu ctg 2465 55 50 7 cta 5 2 2 4 12 ctt 24 4 7 10 19 ctc 1 13 30 10 4 ttg 16 16 510 16 tta 20 1 1 13 42 gly ggg 16 12 16 15 19 gga 31 4 5 11 34 ggt 18 139 34 36 ggc 34 71 70 30 11 val gtg 26 66 47 37 8 gta 20 4 5 15 29 gtt 285 6 26 53 gtc 26 25 42 22 10 ala gcg 26 38 46 36 11 gca 28 13 8 21 42gct 25 5 4 16 34 gcc 21 44 43 27 13 thr acg 27 26 34 27 15 aca 41 5 3 1347 act 16 5 3 17 27 acc 16 63 60 44 11 pro ccg 43 62 59 52 9 cca 19 9 319 33 cct 29 6 4 16 37 ccc 9 23 34 12 13 ile ata 13 1 2 7 45 att 50 16 851 45 atc 37 83 90 42 10 glu gag 32 51 65 31 23 gaa 68 49 35 69 77 aspgat 64 37 35 63 83 gac 36 63 65 37 17 lys aag 30 84 90 23 21 aaa 70 1610 77 79 asn aat 57 28 28 45 78 aac 43 72 72 55 22 cys tgt 45 14 8 45 65tgc 55 86 92 55 35 tyr tat 65 32 61 57 80 tac 35 68 39 43 20 phe ttt 6821 11 57 79 ttc 32 79 89 43 21 gln cag 49 83 91 65 26 caa 51 17 9 35 74his cat 67 39 48 57 84 cac 33 61 52 43 16 met atg 100 100 100 100 100trp tgg 100 100 100 100 100

Values shown reflect the occurrence as a percentage, for each codon, ofthe total codons that specify the given amino acid that the codonencodes. Codon usages were calculated from the genome of B. subtilisstrain 168, X. campestris pv. campestris ATCC33913, E. coli K-12 W3110and Sphingomonas elodea 53 CDSs containing 20,972 codons.

The codon usage shown below in Table 3B is the consensus codon usagetable used in the design of the synthetic genes and the final codonusage of the synthetic gene set as constructed below. The actual usagein the synthetic genes reflects design considerations such as DNA andmRNA secondary structure, inclusion and exclusion of restriction sites,and overall GC content.

TABLE 3B CONSENSUS CODON USAGE Amino Design Synthetic Synthetic acidcodon table, %¹ genes, %¹ genes, #² arg cgg 15 15 52 cga 0 0 0 cgt 25 2688 cgc 60 59 200 agg 0 0 0 aga 0 0 0 ser tcg 30 28 143 tca 0 0.2 1 tct 00.4 2 tcc 25 26 132 agt 0 0 0 agc 45 45 225 leu ctg 70 66 481 cta 0 <0.32 ctt 0 <0.2 1 ctc 15 17 125 ttg 15 16 117 tta 0 <0.2 1 gly ggg 20 17 61gga 0 0 0 ggt 30 33 117 ggc 50 50 176 val gtg 65 60 245 gta 0 <0.3 1 gtt0 0 0 gtc 35 40 162 ala gcg 40 38 169 gca 20 26 118 gct 0 0 0 gcc 40 36161 thr acg 35 39 126 aca 0 0 0 act 0 0 0 acc 65 61 200 pro ccg 70 70194 cca 0 0 0 cct 0 0 0 ccc 30 30 84 ile ata 0 <0.2 1 att 30 31 163 atc70 69 365 glu gag 40 37 154 gaa 60 63 259 asp gat 50 50 186 gac 50 50184 lys aag 50 53 240 aaa 50 47 217 asn aat 40 41 162 aac 60 59 231 cystgt 20 19 18 tgc 80 81 78 tyr tat 40 42 131 tac 60 58 184 phe ttt 34 30101 ttc 66 70 197 gln cag 75 67 171 caa 25 33 84 his cat 50 42 62 cac 5058 86 met atg 100 100 147 trp tgg 100 100 83 ¹Values are % of totalcodons for that amino acid ²Values are total numbers of codons insynthetic gene set as synthesized

A synthetic gene set was constructed as three segments, kpsFEDUCS (the“FS segment”), kpsMTkfoABCFG (the “MG segment”) and kfoDIEH (the “DHsegment”). FIG. 6 diagrammatically presents the structure of these threesynthetic segments. Unique restriction sites, shown in FIG. 6 wereincorporated at strategic locations to allow assembly of the syntheticfragments into one or more operons which can be inserted into plasmidexpression vectors. The initial strategy was to assemble the genes as asingle operon for expression experiments. Other restriction sites alsowere strategically located throughout the synthetic sequences inpositions that allow construction of non-polar deletions for any givengene(s). This can facilitate genetic analysis of the functions of theproteins encoded by the K4 capsule cluster as well as othermodifications of the plasmid sequences. A consensus strong ribosomebinding site (AGGAGGttaataaATG, SEQ ID NO:46) was employed for most ofthe synthetic genes; all except for kpsC, kpsT, kfoE, and kfoH. In theE. coli K4 U1-41 sequence, the translational start sites of these genesare coupled to the translational stops of the genes immediately upstreamand as a result the ribosome binding sites overlap coding sequences ofthose upstream genes.

The synthetic sequences comprising FS, MG and DH segments as definedabove were synthesized as three separate fragments by a commercialvendor, DNA2.0 (Menlo Park. CA). The nucleotide sequences of the threesynthetic segments are listed as: FS segment (SEQ ID NO:47), MG segment(SEQ ID NO:48) and DH segment (SEQ ID NO:49).

Example 3 Construction of Alternative Host Strains

The initial alternate hosts chosen for expression of the K4 biosyntheticgenes included E. coli K-12 (“K-12”), E. coli. B (“EcB”), Sphingomonaselodea (“Sph”), and Xanthomonas campestris pv. campestris (“Xcc”). K-12strains W3110 and MG 1655 were obtained from the Coli Genetic StockCenter, Yale University. Sph strain ATCC 31461 was obtained from ATCC.Xcc strain NRRL B-1459 (ATCC 13951) was obtained from the ARS CultureCollection (NCUAR), Peoria, Ill. E. coli B (ATCC 11303) was obtainedfrom ATCC.

In general, the alternate hosts were prepared for introduction of the K4genes in two ways. It can be advantageous to be able to delivergenes/plasmids to certain alternate hosts by conjugal transfer fromlaboratory cloning strains of E. coli in tri-parental crosses with E.coli containing the mobilizing plasmid pRK2013. To select fortransconjugants of the alternate hosts among the conjugal milieu,antibiotic-resistant derivatives (typically streptomycin resistance) ofthe alternate hosts are required. Alternatively, E. coli strain S17-1(Simon et al., BioTechnology 1983; 1:784-791) can be used. This strainhas a chromosomally integrated form of plasmid RP4 and will directlymobilize appropriate plasmids to new hosts. This strain is streptomycinresistant, however, so streptomycin cannot be used to select againstthis strain among the exconjugants.

Generation of the gene or gene cluster deletions in Sph, Xcc, and K-12was carried out using a two-step, “pop-in/pop-out” homology-drivenmethod (See FIG. 7A). In the first step, a plasmid containing a clonedversion of the desired deletion structure (homologous regions flankingthe deletion) was integrated into the chromosome (popped-in) byrecombination in one of the flanking regions (and selection for a markeron the vector) creating duplications of the homologous flanking regions.In the second step, recombination occurred in the opposite flankingregion removing (popping-out; “resolving”) the cloning vector (plusmarker) and the targeted chromosomal region, but leaving the designeddeletion. Such strains were obtained by multi-generational growth in theabsence of marker selection, followed by screening for loss of themarker and the desired phenotype (determined by colony morphology and/orPCR). For Gram-negative organisms other than E. coli, the desireddeletion is typically built in a “suicide” vector that can be conjugallytransferred to, but cannot replicate in, the target (non-E. coli) hoststrain. For our purposes, we created a “suicide” vector by modifyingpCM184 (see FIG. 7B; Marx and Lidstrom, BioTechniques 2002;33(5):1062-1067). The kanamycin-resistance gene and flanking loxP siteswere removed by digestion with NotI and SacII, polishing of the endswith T4 DNA polymerase and ligation. The resulting plasmid, pCX027 (SEQID NO:141), as shown in FIG. 7B, contained tetracycline-resistance forselection of integrants (in Sph or Xcc) plus a large multi-cloning site.For creating deletions in E. coli, plasmid pMAK705 (Hamilton et al., J.Bacteriol. 1989; 171 (9):4617-4622) was used. This plasmid contained atemperature-sensitive pSC101 replicon so that production of the firststep integration and loss of plasmid sequences in the second(“resolution”) step were facilitated at high temperature. The genestructure of “extracellular polysaccharide (EPS) minus” mutants wasconfirmed by PCR and Southern blot analysis.

The approach to creating deletion constructs was the same for alltargets described above. Upstream and downstream regions of homologywere derived by PCR using the appropriate genomic DNA as templates.Restriction sites were designed into the PCR primers such that theresulting DNA fragments could be cloned into the desired plasmid, or, asfor the Xcc gumD gene described below, restriction sites occurringnaturally in the genome were used. The deletions (and the restrictionsites used for cloning) were designed such that in-frame fusions werecreated between short regions of the N-terminal and C-terminal codingregions of the target gene(s). In effect, the targeted coding regionswere replaced with a restriction enzyme recognition sequence. Theengineered restriction site sequences between the upstream anddownstream fragments added 2-3 non-native codons to the fused codingregions. This procedure yielded well-defined mutations with theexpectation of little/no polar effects on the expression of downstreamgenes.

E. coli K-12

Colanic acid (M antigen) is an extracellular polysaccharide produced bymany enteric bacteria (Grant, W. D., et al., J. Bacteriol. 1969;100:1187-1193), and greater production is typically found at lowergrowth temperatures (Stout, V., J. Bacteriol. 1996; 178:4273-4280). Thecreation of E. coli K-12 strains defective in the colanic acidbiosynthesis is described in this Example. Such strains do not produceinterfering or contaminating colanic acid when they are furtherengineered for the production of recombinant chondroitin which can becarried out at 30° C. or lower. Plasmid pMAK705 (Hamilton, C. M., etal., J. Bacteriol. 1989; 171:4617-4622) was used to create precisedeletions in the chromosomal colanic acid biosynthetic gene cluster.This plasmid contains a temperature sensitive replicon and cannot existin an extra-chromosomal state at higher temperatures. In general, thesteps for creating precise mutations at a target locus result from a“pop-in/pop-out” mechanism and are described by Hamilton et al.(supra.). Plasmid clones containing the designed mutations are driveninto the chromosome (“pop-in”), typically via homologous recombinationat the target locus, by growth of transformants at the non-permissivetemperature and selection for plasmid-encoded antibiotic(chloramphenicol; Cm) resistance. Subsequent multi-generational growthof these integrants in the absence of chloramphenicol results in asub-population of cells in which the plasmid has recombined out of thechromosome and been lost from the cell during cell division leavingbehind either the original wild type structure or the mutant structure.These “pop-outs” are identified by Cm sensitivity. A strain with thedesired mutant structure is then identified by phenotype (if possible)and by PCR or Southern blotting.

The colanic acid biosynthetic operon in E. coli K-12 consists of 19 or20 contiguous genes covering about 24 kb (Stevenson, G., et al., J.Bacteriol. 1996:178:4885-4893). The twentieth gene, wcaM, appears to bepart of the operon/transcription unit but is not required for colanicacid production. Contained therein is the wcaJ gene encoding theglycosyltransferase enzyme responsible for loading of the first sugaronto a lipid carrier during colanic acid biosynthesis. This Exampledescribes the creation of E. coli K-12 strains containing deletions ofeither the entire 20-gene operon or just the wcaJ gene. The sequence ofthe genome of E. coli K-12 strain W3110 is published as GenBank AP009048(Blattner, F. R., et al., Science 1997; 277:1453-1462).

Deletion of the Colanic Acid Operon:

PCR primers were designed to amplify approximately 950 bp upstream ofthe first gene (wza) and downstream of the last gene (wcaM) of the W3110colanic acid operon. These fragments provided homology to the desiredrecombination sites in the chromosome. The PCR primers were designed toresult in non-native restriction sites at the ends of the amplified PCRproducts to be used in subsequent cloning. The upstream region wasamplified with primers CAX129 (HindIII site underlined) and CAX128 (AscIsite underlined). The downstream region was amplified with primersCAX130 (AscI site underlined) and CAX131 (XbaI site underlined).

(SEQ ID NO: 50) CAX128 GGCGCGCCAGCGTCCTGCTGTTTGATGACG (SEQ ID NO: 51)CAX129 AAGCTTGCCAGGAGATTGACGCCAGC (SEQ ID NO: 52)CAX130 GGCGCGCCGGAATCCTCAGTTGGACCCGC (SEQ ID NO: 53)CAX131 TCTAGAACTTTACCCTCACGGTCCAGCG

PCR was performed with Pfu polymerase (Stratagene) for 30 cycles ofdenaturation at 95° C., annealing at 57° C. and extension at 72° C. (20sec. each step). The template consisted of 100 ng of E. coli K-12 W3110genomic DNA. The PCR fragments were cloned into pCR-Blunt II-TOPO(Invitrogen) and transformed into E. coli TOP10 (Invitrogen), and thesequences of selected cloned inserts were confirmed to match publisheddata. An upstream clone was digested with HindIII plus AscI, and adownstream clone was digested with XbaI plus AscI. The desired fragmentswere gel-purified (Gene Clean Turbo, Q-BIOgene) and ligated with pMAK705that had been digested with HindIII plusXbaI followed by treatment withAntarctic phosphatase (New England Biolabs). Cm-resistant (LB Cm 34μg/mL, 30° C.) transformants of E. coli DH5α (Invitrogen) were analyzedfor plasmid structure, and one with the desired structure was namedpMAK-CL. In this plasmid (and the chromosomal deletion ultimatelyderived from it), the union of the upstream and downstream fragments atthe AscI site resulted in a small 345 bp open reading frame consistingof the 5′ end of the wza gene and the 3′ end of the wcaM gene. Thisfeature was designed to minimize disruption of potential expression fromthe colanic acid operon promoter of any genes farther downstream ofwcaM.

Plasmid pMAK-CL was transformed into E. coli W3110 by electroporationwith selection for Cm resistance by plating on LB (Maniatis, 1989) agarplates containing Cm at 17 g/mL at 30° C. (permissive temperature).Several transform colonies were streaked to M9 (Maniatis, 1989) Cm 17μg/mL agar plates and incubated at 43° C. (non-permissive temperature).After two days, multiple colonies (presumptive integrants) were present,and these were re-streaked to M9 Cm at 43° C. for confirmation. Twopresumptive integrants were grown for about 25 generations in LB mediumat 37° C., and dilutions were prepared and spread to LB plates forgrowth at room temperature (22-24° C.). After three days, colonies weretransferred to two LB agar plates, one containing Cm 17 μg/mL, forgrowth at 30° C. Cm-sensitive isolates were present at frequencies of62% and 94% among derivatives of the two original integrants. Thesepresumably resulted from “pop-out” and loss of the pMAK-CL plasmid.“Colony PCR” was used to evaluate the structure at the colanic acidoperon in these strains. A small amount of a colony was suspended in 10μL of sterile de-ionized water in a tube compatible with PCR. To thiswas added 20 μL of a 1.5-fold concentrated mix of “Taq Master”(Eppendorf) components such that the final concentrations/amounts in thereaction were: 1× Taq polymerase buffer, 1× “Taq Master” reagent, 0.33mM each dNTP, 0.4 μM each primer, and 0.5 units Taq polymerase. PCR wasstarted with 8 minutes at 95° C., continued with 35 cycles ofdenaturation at 95° C. for 30 sec., annealing at 55° C. for 30 sec., andextension at 68° C. for 3 min., and finished with extension at 68° C.for 7 min. Initial characterization was carried out with a forwardprimer (CAX132) in the upstream homologous region and a reverse primer(CAX135) in the downstream homologous region.

(SEQ ID NO: 54) CAX132 CCGAATTGTTATCTTGCCTGC (SEQ ID NO: 55)CAX135 GGTAGCATCTCTTTGGGTATCG

PCR of strains containing the desired operon deletion were expected toproduce a 1000 bp fragment, and this was found in 9 of 23 “pop-out”strains analyzed. To ensure that undesired rearrangements had notoccurred in these strains, “colony PCR” with primers outside of theregions of homology were used: CAX162 (forward) and CAX163 (reverse).

(SEQ ID NO: 56) CAX162 GAACAGCGGTTGAGTCAGGG (SEQ ID NO: 57)CAX163 GGCAGAAAGCACATAGCGACC

These outside primers gave a PCR product of 2065 bp in deletions of thedesired structure, and 4 of the 9 strains produced this PCR product; oneof these was designated MSC188. Further confirmation of the structure ofthe deletion in strain MSC188 was achieved by Southern blotting. A“DIG”-labeled probe (Roche) was generated with primers CAX128 and CAX129(1000 bp) using pMAK-CL as template. Chromosomal DNA from wild type E.coli W3110 and MSC188 was digested with restriction enzymes KpnI, PstIand BglII, and the digests were subjected to gel electrophoresis andblotting. Probing revealed the expected band patterns in MSC188 andW3110, respectively: KpnI, 5921 bp vs. 9431 bp; PstI, 3902 bp vs. 12893bp, and BglII, 9361 bp vs. 6201 bp.

Deletion of wcaJ:

The strategy for deletion of the wlaJ gene in E. coli K-12 W3110followed that described above for deletion of the entire colanic acidbiosynthetic operon. PCR primers were designed to amplify approximately500 bp upstream and downstream of the wcaJ gene of the W3110 colanicacid operon. The upstream region was amplified with primers CAX126(HindIII site underlined) and CAX125 (PacI site underlined). Thedownstream region was amplified with primers CAX124 (PacI siteunderlined) and CAX127 (XbaI site underlined).

CAX124 (SEQ ID NO: 58) TTAATTAACAAAGGTTTCGTTAACAAAGCGG CAX125(SEQ ID NO: 59) TTAATTAAATTGGTTTTCGCTCGCTCGC CAX126 (SEQ ID NO: 60)AAGCTTGGAAGACGCCATCTATGGTGG CAX127 (SEQ ID NO: 61)TCTAGAGAAGCCCGCCAGCACCGC

Restriction fragments of the upstream and downstream PCR products werecloned into pMAK705 to yield pMAK-wca. In this plasmid and thechromosomal deletion ultimately derived from it, the union of theupstream and downstream fragments at the PacI site resulted in a small75 bp open reading frame consisting of the 5′ and 3′ ends of the wcaJgene. This feature was designed to allow un-interrupted expression ofall other operon genes in the event that they benefit production ofchondroitin. Initial characterization of presumptive wcaJ deletionderivatives of E. coli W3110 by colony PCR was carried out with primersCAX126 and CAX127, and 11 of 23 prospective “pop-outs” gave the desiredsignal. Outside primers CAX160 (forward) and CAX161 (reverse) were usedto identify presumptive wcaJ deletions, and 3 of 4 strains testedcontained the expected product (1188 bp). One strain with the desiredDNA structure was designated MSC175.

CAX160 (SEQ ID NO: 62) CCGTTGATGTGGTGACTGCC CAX161 (SEQ ID NO: 63)AAACAGCAGCGTTCTCACCG

For Southern blot confirmation, the “DIG”-labeled probe was generatedwith primers CAX124 and CAX127 (514 bp) using pMAK-wca as template.Chromosomal DNA from wild type E. coli W3110 and MSC175 was digestedwith restriction enzymes PacI, DraIII and NdeI, and the digests weresubjected to gel electrophoresis and blotting. Probing revealed theexpected band patterns in MSC175 and W3110, respectively: PacI, 8456 bpvs. >28000 bp; DralII, 4502 bp vs. 5819 bp, and NdeI, 8512 bp vs. 9829bp.

Xanthomonas Campestris

Xanthomonas campestris pv. campestris (Xcc) is used commercially toproduce the extracellular carbohydrate polymer xanthan gum for a varietyof industrial and food applications (Baird, J., et al., BioTechnology1983; 1:778-783). In order to employ this strain and process forproduction of chondroitin, Xcc strains unable to biosynthesize xanthangum are required. A strategy similar to that used (above) for E. coliwas used to delete the entire xanthan gum biosynthetic operon or justthe gene for the first glycosyltransferase, gumD, in Xanthomonascampestris pv. campestris strain ATCC 13951, also known as NRRL B-1459(Capage, M. R. et al., World Patent WO87/05938; Katzen, F., et al., J.Bacteriol. 1998; 180(7):1607-1617). First, a spontaneously-arisingderivative resistant to 100 μg/mL streptomycin sulfate in nutrient agarat 30° C. was obtained and named MSC116. PCR primers were designedagainst the sequence of the xanthan gum biosynthetic cluster of strainNRRL B-1459 (GenBank accession #U22511) and, where necessary, to thegenomic sequence for X. campestris pv. campestris ATCC33913 (GenBankaccession AE008922).

Deletion of gumD:

The strategy for deletion of the gumD gene takes advantage of naturallyoccurring EcoRI restriction sites approximately 1650 bp upstream (5′)and 1000 bp downstream (3′) of the coding region. PCR primers weredesigned outside of these restriction sites, and these were paired withPCR primers targeting regions just inside of the gumD coding sequence togenerate upstream and downstream regions of homology. To amplifyapproximately 1800 bp of upstream homology, primers CAX114 and CAX116were used. To amplify approximately 1100 bp of downstream homology,primers CAX115 and CAX117 were used. The two middle primers in the endsof the gumD coding region were amended with SbfI restriction sites(underlined below).

CAX114 (SEQ ID NO: 64) CCTGCAGGGTCGAACACTCGCAAGACCAGG CAX115(SEQ ID NO: 65) CCTGCAGGTATCCGCATCATCGTGCTGACG CAX116 (SEQ ID NO: 66)CCTTGGTGATGGTGTGGCG CAX117 (SEQ ID NO: 67) GCCCATCCACGACTCGAACG

PCR was performed with Pfu Ultra II polymerase (Stratagene) for 30cycles of denaturation at 95° C. (20 sec.), annealing at 62° C. (20sec.) and extension at 72° C. (30 sec.). Template consisted of 100 ng ofX. campestris pv. campestris (strain ATCC13951; “Xcc”) genomic DNA. ThePCR fragments were cloned into pCR-Blunt II-TOPO (Invitrogen) andtransformed into E. coli TOP10 (Invitrogen). The relevant sequence of anupstream homology clone matched the sequence of the PCR product, butthis shared sequence differs by two base pairs from the published Xccsequence for this region (primers excluded). It is likely that thepublished sequence contains a small number of incorrect assignments. Therelevant sequence of a downstream homology clone matched the sequence ofits PCR product and that of the published Xcc sequence for this region(primers excluded).

The strategy for creating specific gene deletions using a“pop-in/pop-out” mechanism in Xanthomonas was based on a derivative ofplasmid pCM184 (Marx, C. J., and Lidstrom, M. E., BioTechniques33(5):1062-1067, 2002). Plasmid pCM84 consisted of a ColEI replicon,which permits replication of the plasmid in E. coli but not inXanthomonas (or other non-enteric bacteria), an oriT region for conjugaltransfer among Gram negative bacteria, resistance genes for ampicillinand tetracycline, plus a resistance gene for kanamycin (Kan^(r)) flankedby loxP sequences. This plasmid was designed to create unmarked (noremaining antibiotic resistance gene) deletions in non-E. coli strains,but the described procedure left the small loxP sequence at the site ofthe deletion. However, it was desirable for the strains created in thepresent invention to contain no unnecessary sequences such as loxP.Therefore, pCM184 was modified to remove the loxP sites and theintervening kanamycin resistance gene. This allowed for use of theresulting derivative plasmid for creation of desired unmarked deletionsin Xanthomonas by a variation on the “pop-in/pop-out” mechanismdescribed above.

About 1.8 μg of plasmid pCM184 was digested with NotI plus SaclIrestriction enzymes (chosen to remove the loxP/Kan^(r)/loxP region butleave most restriction sites for future use), and the completed reactionwas heated to 75° C. for 15 min. to inactivate the enzymes. The sample(20 μL) was then treated with T4 DNA polymerase (1.8 U, New EnglandBiolabs) plus 100 μM each dNTP for 15 min. at 12° C. to fill-in thesingle strand overhang from NotI digestion and to trim back the overhangfrom SacII digestion (i.e., create blunt ends). Reaction was terminatedby the addition of EDTA to 10 mM and heating at 75° C. for 20 min. About170 μg of treated plasmid was reacted with 400 U T4 DNA ligase (NewEngland Biolabs) in a 10 μL volume at 16° C. for 4 hrs. The ligationreaction was subsequently treated with 2.5 U SbfI for 90 min to digestunwanted DNA structures (such as reformed pCM184). A 0.5 μL volume ofthis reaction was used to transform E. coli TOP 10 (Invitrogen), andplating to LB Tc⁵ resulted in numerous colonies after overnightincubation at 37° C. The plasmid contained in cells from a chosen colonywas shown by restriction enzyme analysis and DNA sequencing to be of thedesired structure: this plasmid was named pCX027 (SEQ ID NO: 141) and isdiagrammed in FIG. 7B.

The plasmids (about 2 μg each) containing the cloned PCR products forthe gumD upstream and downstream regions (above) were digested with 10 USbfI for 2 hr at 37° C., then 10 U EcoRI (with enzyme-specific buffer)under the same conditions. After heat treatment (70° C. for 20 min.),the digests were subjected to agarose gel electrophoresis, and thedesired fragments (upstream fragment about 1.6 kb; downstream fragmentabout 1.1 kb) were purified with the QIAGEN Mini-Elute kit. PlasmidpCX027 (about 3.5 μg) was digested with 20 U EcoRI in a 50 μL reactionfor 2.5 hr at 37° C., treated with about 18 U Antarctic phosphatase (NewEngland Biolabs) in a 60 μL reaction for 15 min. at 37° C., and thenheated at 70° C. for 20 min. A three-way ligation was carried out withabout 100 ng each of treated pCX027 and purified gumD upstream anddownstream fragments in 10 μL reactions at 16° C. for about 20 hr. Halfof this reaction was transformed into E. coli DH5α (Stratagene) followedby plating to either LB Ap¹⁰⁰ or LB Tc⁵ at 37° C. Colony PCR wasperformed (as described above) to identify clones with the desiredstructure using primers CAX122 and CAX119 homologous to regions in theupstream and downstream sequences, respectively.

CAX119 (SEQ ID NO: 68) GACCAATGACACGATGATCG CAX122 (SEQ ID NO: 69)GCATCCGCTACAACATGCTC

PCR products of the expected size (1169 bp) were detected in severalcolonies, and the desired structures were confirmed by restrictionanalysis. Plasmids in which the orientation (given as the gum genereading frames) of the paired homology regions were the same or oppositeof that for the vector Tet^(r) gene were named pCX030 and pCX031,respectively.

Xanthomonas campestris pv. campestris (“Xcc”; B-1459 from the ARSCulture Collection (NCUAR) Peoria, Ill.; also known as ATCC13951) wasgrown in nutrient broth (NB, Difco) overnight at 30° C. (all growth ofXcc strains at 30° C. unless indicated), diluted 1:5 in fresh NB, and100 μL aliquots were spread to nutrient agar (NA, Difco) platescontaining streptomycin (str) at 100 μg/mL followed by incubation at 30°C. After several days, colonies were detected at a frequency of about 1per 10⁷ originally-plated cells. Several spontaneously-arisingstreptomycin-resistant Xcc strains were purified by streaking to NAstr⁵⁰ plates, and one such isolate was named MSC116.

Plasmid pCX030 or pCX031 was transferred to Xcc strain MSC116 byelectroporation (Oshiro et al., J. Microbiol. Method 2006; 65:171-179).Tetracycline-resistant colonies (TC^(R)) were obtained fromtransformants from pCX030 (4.1×10⁴/μg) and pCX031 (3.1×10⁴/μg),respectively. Genomic DNA was prepared from isolated Tc^(R) strainsderived from pCX031 transformation and evaluated by PCR for the site ofpCX031 integration. Primer pairs were chosen to determine linkage ofgenomic sequence outside of the upstream region used in pCX031 to thedownstream region and sequence outside of the downstream region used inpCX031 to the upstream region. Specifically, primer CAX116 (“outsideprimer” for upstream region) plus CAX119 (in downstream homologousregion; see above) were used to test the upstream linkage and CAX117(“outside primer” for downstream region; see above) plus CAX122 (inupstream homologous region; see above) were used to test the downstreamlinkage. PCR was conducted with Go Taq DNA polymerase (Promega, Madison,Wis.) with 0.5 μM each primer, 250 μM each dNTP, 1000 ng DNA template,and 0.5 U enzyme. Reaction conditions include initial denaturation at94° C. for 4 min., 30 cycles of 15 sec. denaturation at 94° C., 30 sec.annealing at 55° C., and 4 min. extension at 72° C. and final extensionfor 2 min. “Pop-in” isolates were identified for both upstream anddownstream integration of pCX031. Two isolated “pop-in” strains weredesignated MSC221 and MSC222. These strains were inoculated in LBLS (10g/L Bacto peptone, 5 g/L NaCl, 5 g/L yeast extract, no antibiotics) forgrowth at 30° C., and then thrice sub-cultured into the same medium at48 hr intervals using 1:1000 dilutions. The resulting cultures werediluted, and aliquots were spread to NA Str⁵⁰ plates. Resulting colonieswere transferred to NA and NA Tc⁵ plates. Tc⁵ strains were found at afrequency of 2% from both strains. Colony PCR analysis against selectedTc⁵ strains using the primer pairs (CAX116 plus CAX119 and CAX117 plusCAX122), as described above, demonstrated that all strains tested wereconsistent with gumD deletion. These isolated “pop-out” strains fromMSC221 and MSC222 were designated MSC225 and MSC226, respectively.Colonies of MSC225 and MSC226 on agar plates were clearly non-mucoidrelative to colonies of the MSC116 parent strain.

Deletion of the Xanthan Gum Biosynthetic Gene Cluster:

Deletion of the gumB thru gumM biosynthetic cluster largely follows thesame steps detailed for deletion of the gumD gene. Regions of homologyupstream of gumB and downstream of gumM were created by PCR with primersCAX136×CAX137 (1434 bp) and CAX138×CAX139 (1420 bp), respectively,incorporating a BglIII restriction site for cloning into pCX027 and NotIsites for fusion between the upstream and downstream fragments. Fusionof the NotI sites will create an open reading frame for a 53 amino acidpolypeptide consisting of the 5′ end of the gumB and 3′ end of the gumMcoding sequences. Restriction sites are underlined: BglII in CAX136 andCAX139; NotI in CAX137 and CAX138.

CAX136 (SEQ ID NO: 70) AGATCTGGCGGTAACAGGGGATTGGC CAX137 (SEQ ID NO: 71)GCGGCCGCCAAGACGGTATTCGGGCTGC CAX138 (SEQ ID NO: 72)GCGGCCGCGATCTGCTGGTGTTCTTCCGC CAX139 (SEQ ID NO: 73)AGATCTCCTACCGACCAGGCATTGGC

PCR was performed with Pfu Ultra II polymerase (Stratagene) foramplification of upstream and downstream fragments. Reaction conditionsinclude initial denaturation at 94° C. for 4 min., 30 cycles of 20 sec.denaturation at 95° C., 30 sec. annealing at 57° C., 30 min. extensionat 72° C. and final extension at 72° C. for 5 min. Template consisted of100 ng of X. campestris pv. campestris (strain ATCC13951; “Xcc”) genomicDNA. The PCR fragments were cloned into pCR-Blunt II-TOPO (Invitrogen)and transformed into E. coli TOP10 (Invitrogen). The relevant sequenceof an upstream homology clone matches the sequence of the PCR product,but this shared sequence differs by 14 base pairs from the publishedsequence of Xcc strain ATCC33913 for this region (primers excluded).These sequence variances likely reflect subtle differences between theB-1459/ATCC13951 and ATCC33913 genomes. The relevant sequence of adownstream homology clone matches the sequence of the PCR product andthis sequence does not differ from the published Xcc ATCC33913 sequencefor this region (primers excluded). The plasmids (about 1 μg each)containing the cloned PCR products for the upstream region of gumB(above) and the downstream region of gumM (above) were digested with 7.5U NotI and 7.5 U BglIII for 2 hr at 37° C. The digests were subjected toagarose gel electrophoresis, and the desired fragments (each about 1.4kb) were purified with the QIAGEN Mini-Elute Kit. Plasmid pCX027 (about1.0 μg) was digested with 15 U BglII in a 15 μL reaction for 2 hr at 37°C., treated with about 5 U Antarctic phosphatase (New England Biolabs)in a 75 μL reaction for 15 min. at 370, and then heated at 65° C. for 10min. After purification of BglII digested pCX027, a three-way ligationwas carried out with purified pCX027, the upstream fragment of gumB, andthe downstream fragment of gumM in 20 μL reactions at room temperaturefor 3 hr. Half of the reaction mixture was transformed into E. coliTOP10 (Invitrogen) followed by plating to either LB Ap¹⁰⁰ or LB Tc⁵ at37° C. Colony PCR was performed (as described above) to identify cloneswith the desired structure using CAX140 (in upstream homologous region)and CAX145 (in downstream homologous region).

CAX140 (SEQ ID NO: 74) CCGAATTTCCGAGCCTGG CAX145 (SEQ ID NO: 75)GCCCGCTCGCTTCGTCG

Plasmid was prepared from PCR positive clones with the QIAGEN QiaprepSpin Miniprep Kit and digested with BglII, NdeI or NcoI to confirm thestructure of the plasmid including its orientation of homologous regionsin the upstream and downstream sequences. Plasmids in which theorientations of the paired homology regions were the same or opposite ofthat for the vector Tet^(r) gene were designated pKM001 and pKM002,respectively.

Plasmid pKM001 or pKM002 was transferred to Xcc strain MSC116 byelectroporation (Oshiro et al., J. Microbiol. Method 2006; 65:171-179).Tc^(R) colonies were obtained from transformants of pKM001 (5.3×10³/μg)and pKM002 (5.0×10³/μg), respectively. Genomic DNA was prepared fromisolated Tc^(R) strain derived from pKM001 transformant and evaluated byPCR for the site of pKM001 integration. Primer pairs were chosen todetermine linkage of genomic sequence outside of the upstream regionused in pKM001 to the downstream region and sequence outside of thedownstream region used in pKM001 to the upstream region. Specifically,primer prKM001 (“outside primer” for upstream region; see below) plusCAX145 (in downstream homologous region; see above) were used to testthe upstream linkage and prKM002 (“outside primer” for downstreamregion; see below) plus CAX142 (in upstream homologous region; seebelow) were used to test the downstream linkage.

prKM001 (SEQ ID NO: 76) ACGTGGATGCGGTCGTCGC prKM003 (SEQ ID NO: 77)GGGGCTTGCGGGTCGGC CAX142 (SEQ ID NO: 78) CGTATGCTGAGAATGACGACC

PCR was conducted with Go Taq DNA polymerase (Promega) with 0.5 μM eachprimer, 250 μM each dNTP, 600-1000 ng DNA template, and 0.5 U enzyme.Reaction conditions include initial denaturation at 94° C. for 5 min.,30 cycles of 15 sec. denaturation at 94° C., 30 sec. annealing at 55°C., and 4 min. extension at 72° C. and final extension for 2 min.“Pop-in” isolates were identified for upstream integration of pKM001.These isolated strains were designated MSC242, MSC247 and MSC248.

MSC242, MSC247 and MSC248 were inoculated in LBLS medium for growth at30° C., and then thrice sub-cultured into the same medium at 48 hrintervals using 1:1000 dilutions. The resulting cultures were diluted,and aliquots were spread to NA Str⁵⁰ plates. Resulting colonies weretransferred to NA and NA Tc⁵ plates. Tc^(S) strains were observed atfrequencies of 1-2% from the three strains. Genomic structure inselected Tc^(S) strains was evaluated by PCR to confirm deletion ofxanthan gum synthesis genes from gumB to gumM using prKM001 plus CAX145for upstream linkage and prKM003 plus CAX142 for downstream linkage (seeabove). PCR was conducted with Herculase II Fusion DNA polymerase(Stratagene) with 0.25 μM each primer, 250 μM each dNTP, 500-700 ng DNAtemplate, and 0.5 U enzyme. Reaction conditions included initialdenaturation at 98° C. for 4 min., 30 cycles of 20 sec denaturation at98° C., 20 sec. annealing at 60° C., and 2 min. extension at 72° C. andfinal extension for 4 min. Three “pop-out” strains (one from each“pop-in” strain) showed the PCR products consistent with deletion of thexanthan gum biosynthetic gene cluster. These xanthan gum biosynthesisgene deletion “pop-out” strains from MSC242, MSC247 and MSC248 weredesignated MSC255, MSC256 and MSC257, respectively. Colonies of MSC255,MSC256, and MSC257 on agar plates were clearly non-mucoid relative tocolonies of the MSC116 parent strain.

E. coli B

The genome of E. coli BL21 (DE3), a derivative of wild type E. coli B(ATCC11303), was reported to contain an inactive group 2 capsule genecluster in which the regions 1 and 3 were intact (and functional), butregion 2 was disrupted and non-functional (Andreishcheva, E. N., andVann, W. F., Gene 2006; 384:113-119). Given that genes of regions 2 werepolymer-specific but that regions 1 and 3 were generic and lessspecific, E. coli B can be engineered to synthesize chondroitin byproviding just the K4 region 2 genes on a plasmid or integrated into thechromosome (see below). To improve the utility of E. coli B as a hostfor chondroitin production, the production of colanic acid waseliminated by genetic mutation as described for E. coli K-12 above.

Deletion of the Colanic Acid Operon:

The process for deletion of the E. coli B colanic acid operon followsthat used for the K-12 strain described above. At the time of thisinvention, the E. coli B genome sequence was not publicly available.Though the K-12 and B strains are closely related, some differences inDNA sequences are expected. Therefore, creation of new upstream anddownstream homologous regions was needed, and the existing primers usedfor strain K-12 were employed. Specifically, PCR with primers pairsCAX128×CAX129 and CAX130×CAX131 and E. coli B genomic DNA template wasused to generate upstream and downstream homologous regions,respectively. Products of about 1 kb in size were obtained, cloned, andsequenced. In the non-primer sequences, the upstream homologous region(944 bp) differs by only two bases (transitions) from the K-12 upstreamregion, and the downstream homologous regions (911 bp) differs by 30bases (24 transitions, 6 transversions). The upstream and downstreamfragments were cloned into pMAK705 to generate pMAK-BCL.

Plasmid pMAK-BCL was introduced into E. coli B by electroporation. LBmedium was inoculated with a fresh colony and incubated overnight at 37°C. with vigorous shaking. A volume of fresh, pre-warmed LB wasinoculated with the overnight culture to give an initial OD600 readingof 0.03 (BioPhotometer, Eppendorf). The culture was grown to OD600≈0.8and then chilled on ice for 30-40 minutes. Cells were collected bycentrifugation (10 min., 4000 g), and the cells were washed twice byre-suspension in original volumes of ice-cold deionized water followedby re-centrifugation. The cells from the final centrifugation weresuspended in 1/500^(th) volume of ice-cold water. pMAK-BCL (200 ng) wasadded to 50 μL of prepared E. coli B suspension and incubated on ice forabout 20 min. Electroporation was carried out with Gene Pulser Xcell(BioRad) in 0.1 mm gap cuvettes at settings of 25 μF, 200Ω, and 1.8 kVyielding durations of 4.5-5.0 msec. Pulsed cells were diluted with 350μL SOC medium (Maniatis, 1989) and incubated at 37° C. for 1 hr, 5-10 μLwas then spread to LB Cm³⁴ agar plates with incubation at 43° C.Colonies appearing after 2 days (representing “pop-in” candidates) werestreaked to LB Cm³⁴ agar plates at 43° C., and resulting colonies wereinoculated into LB medium (no Cm) for growth and serial passage at 30°C. Colonies derived from these cultures were tested for Cm-sensitivity,and “pop-out” candidates were identified. Colony PCR was used tocharacterize the candidate strains. One isolate was found to give theexpected PCR products using primer pairs CAX129×CAX32, CAX131×CAX132,CAX132×CAX135, CAX129×CAX135, and CAX162×CAX163. This E. coli B isolatedeleted for the colanic acid gene cluster was named MSC364.

Example 4 Construction of Expression Vectors

Well characterized high-copy-number and low-copy-number plasmid vectorsspecific to E. coli have been described (Balbas and Bolivar, MethodsEnzymol. 1990:185:14-37, Das, Methods Enzymol. 1990; 182:93-112,Mardanov et al. Gene 2007; 15(395):15-21). Such vectors employ a varietyof well characterized promoter systems for regulated gene expression inE. coli. Additionally, conjugally-transmissible plasmid vectors based onbroad host range plasmids such as RK2 (low copy number IncP) and RSF1010(high copy number IncQ) that function in E. coli, X. campestris and awide variety of other gram negative bacteria are also available(Franklin and Spooner, Promiscuous plasmids in Gram-negative bacteriaAcademic Press (London) 1989 pp 247-267, Mather et al. Gene 1995;15:85-88, Haugen et al., Plasmid 1995; 33:27-39. Mermod et al., J Bact.1986; 167:447-454). The synthetic chondroitin biosynthetic gene set canbe cloned into these versatile broad host range vectors so that the sameplasmids can be used for gene transfer and expression in a wide array ofgram-negative bacteria including X. campestris, S. elodea, P. putida,and non-pathogenic E. coli (Guiney and Lanka, Promiscuous plasmids inGram-negative bacteria Academic Press (London) 1989 pp 27-54).

Many useful IncP-based vectors are derived from the RK2, a conjugallyself-transmissible plasmid originally isolated from clinical Pseudomonasisolates and subsequently shown to be capable of transferring itselfinto, and functioning within, nearly every gram negative bacteriumtested. Smaller derivatives of RK2 have been constructed that are stablereplicons and can be conjugally transferred when “helper” functions aresupplied in trans from a second plasmid. One such plasmid is pFF1(Durland et al., J. Bact. 1990; 172:3859-3867). Some useful derivativesof this plasmid have been described; one of these is pJB653 (Blatny etal., Appl. Enviorn. Micro. 1997; 63:370-379) which adds the Pm promoterof the Pseudomonas TOL plasmid and the regulatory gene xylS to provide astrong, well regulated promoter shown to function in a variety of gramnegative bacteria. This vector and related constructs are the subject ofU.S. Pat. No. 6,258,565. Various IncQ-based plasmid vectors have beenderived from RSF1010, an 8.7 kb plasmid originally isolated fromPseudomonas putida. RSF1010 can propagate in E. coli and a wide varietyof gram negative bacteria. Derivatives of RSF 1010 carrying the Pmpromoter and xyvS regulatory protein have been constructed anddescribed. The plasmid pNM 185 (Mermod et al., J. Bact. 1986;167:447-454) is an RSF 010 derivative that carries the Pm promoter andxylS regulatory gene.

Schumann et al. (Plasmid 2005; 54:241-248) have described series ofplasmid-based expression vectors for Bacillus subtilis that allow stableintracellular expression of recombinant proteins. These expressionvectors are based on the E. coli-B. subtilis shuttle vector pMTLBS72which replicates in B. subtilis as theta circles and is consequentlymore stable than typical B. subtilis plasmids such as pUB110 whichreplicates via a rolling circle mechanism. Derivatives of this plasmidwhich contain constitutive promoter PlepA, promoter PgsiB which can beinduced by heat and acid shock, and by ethanol, and the PxylA and Pspacpromoters which respond to the addition of xylose and IPTG,respectively, have been described.

Broad-host-range plasmid pBHR1 (Szpirer et al., J. Bacteriol. 2001;183:2101-10), which is reported to be compatible with IncP and IncQplasmids, was purchased from MoBiTec GmbH (Goettingen, Germany). Thisplasmid was modified to create a vector (pDD54) employing the Pm/xylSexpression system referenced above. The first step in constructing thepBHR1-based expression vector was to delete the kanamycin resistance(KanR) gene present on that plasmid. This was desirable because pRK2013,a plasmid that can be used to direct conjugal transfer of pBHRa andderivatives, also carried a KanR gene. Moreover, deletion of that gene,and flanking sequences, facilitated certain subsequent cloning stepsdetailed below. pBHR1 also carries a gene that conferschloramphenicol-resistance (CamR) and that antibiotic can be usedinstead of kanamycin to select for this plasmid. Plasmid DNA wasprepared from pBHR1 (diagrammed in FIG. 8A) digested with SbfI and the1.2 kb SbfI fragment containing the KanR gene was deleted by ligatingthe digestion products and screening for chloramphenicol-resistant,kanamycin-sensitive transformants. The plasmid from one suchtransformant was designated pDD39 (see FIG. 8A) and used in furtherconstruction steps.

The xylS gene, which positively regulates expression from the Pmpromoter, was amplified by PCR from pWW0 (TOL plasmid) DNA prepared fromPseudomonas putida ATCC 33015. Using the QIAGEN Plasmid Mini Kit (QIAGENInc., Valencia, Calif.) according to the vendor protocol 4 μg of pWW0DNA was isolated from 20 mL of a fresh overnight culture of Pseudomonasputida ATCC 33015. This DNA preparation was used as template for PCRreactions which amplified the xylS gene and flanking DNA sequences astwo fragments which were subsequently joined together by a subsequentPCR splicing reaction. This procedure facilitated the addition of a NsiIsite 9 base pairs downstream of (3-prime to) the translational stopcodon of xylS. In the initial round of PCR one reaction (Reaction A)employed primers DHD197 (SEQ ID NO:103) and DHD201 (SEQ ID NO:104) and asecond reaction (Reaction B) employed primers DHD200 (SEQ ID NO:105) andDHD198 (SEQ ID NO:106). Sequences of these primers are as follows:

DHD197 (SEQ ID NO: 103) 5>GCACTGCAGATCCCCTTTATCCGCC>3 DHD198(SEQ ID NO: 106) 5>GCACTGCAGATCCACATCCTTGAAGGC>3 DHD200 (SEQ ID NO: 105)5> GATTACGAACGATGCATAGCCGAAGAAGGGATGGGTTG >3 DHD201 (SEQ ID NO: 104)5>CTTCTTCGGCTATGCATCGTTCGTAATCAAGCCACTTCC>3

PCR reactions were performed using PfuUltra II polymerase (STRATAGENE,LaJolla, Calif.) according to the vendor protocols. In each 100 μLreactions, primers were added to a final concentration of 0.4 μM each,dNTPs were added at a final concentration of 200 μM each and 10nanograms of pWW0 DNA was added as template. PCR reactions wereperformed in a Perkin-Elmer GeneAmp 2400 thermocyler using the followingcycling parameters: 1 cycle of 2 min. at 95° C.; 30 cycles of 20 sec. at95° C., 20 sec. at 60° C., and 45 sec. at 72° C.; 1 cycle of 3 min. at72° C.; and a hold at 4° C. Products of these reactions were analyzed byagarose gel electrophoresis. The sizes of PCR products observed wereconsistent with the expected sizes for the products of both Reaction A(1259 bp) and Reaction B (422 bp).

The products of these reactions were purified using the Qiagen QIAquickPCR Purification Kit (QIAGEN, Valencia, Calif.) according to the vendorprotocol, and 1 μL of each was added to 1 mL of sterile distilleddeionized water. To 50 μL of this mixture was added 10 μL of 10×PfuUltra II reaction buffer, 10 μL of a stock solution of dNTPS (10 mMeach), 10 μL of a stock solution of DHD197 (4 μM), 10 μL of a stocksolution of DHD198 (4 μM), 16 μL of sterile distilled deionized waterand 2 μL of PfuUltra II polymerase. The PCR reaction was performed usingthe procedure described above for Reactions A and B. The products ofthis reaction were purified using the Qiagen QIAquick PCR PurificationKit (QIAGEN, Valencia, Calif.) according to the vendor protocol andanalyzed by agarose gel electrophoresis. A strong band was observed at aposition consistent with the expected size of the product of the PCRsplicing reaction, 1610 bp. This band was excised from the gel using theQIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) according tothe vendor protocol.

This PCR fragment was cloned into the pCR-Blunt II-TOPO cloning vector(Invitrogen, Carlsbad, Calif.) according to the vendor protocol. Theresulting plasmid was designated pDD42 and is diagramed in FIG. 8A. ThePCR primers DHD197 and DHD198 added PtI sites 3 base pairs from each endof the 1610 bp PCR fragment. The sequence of the PstI fragment in pDD42was determined (SEQ ID NO:107). This sequence matched the expectedsequence for the xylS gene based on the reported pWW0 sequence (GenBank,AJ344068) and showed the addition of 5 base pairs derived from primersDHD200 and DHD201 which results in the creation of a NsiI site 9 basepairs downstream (3′) of the translational stop codon of xylS. In thenon-coding region downstream of xylS, two sequence differences wereobserved between the cloned PstI fragment in pDD42 and the sequencereported in GenBank, AJ344068. Insertions of a G residue are observed119 and 181 bp 3′ to the TGA stop codon of the xylS gene. These sequencedifferences occur within the intergenic region between xylS and xylHgenes.

The PstI fragment containing the xylS gene was excised from pDD42,gel-purified and cloned into the SbfI site of pDD39. PstI and SbfIenzymes create digestion products having identical 4 bp overhangs, whichcan be ligated together, but the SbfI recognition site is destroyed inthe resulting recombinants. The pDD39 derivative containing the xylSgene contained on the PsiI fragment from pDD42 was termed pDD47 and isshown in FIG. 8A.

A 470 bp DNA fragment (SEQ ID NO:79) that comprises approximately 90 bpof TOL plasmid DNA sequence spanning the minimal Pm promoter sequencesrequired for binding of RNA polymerase and the XylS protein(Dominguez-Cuevas er al., 2008) plus synthetic upstream and downstreamtranscriptional terminators and multiple restriction sites for cloninggenes immediately downstream of the Pm promoter was synthesized de novoby DNA 2.0 (Carlsbad, Calif.). FIG. 8B shows pJ201:11352 which containsthis 470 bp fragment cloned in the DNA 2.0 pJ201 vector. Thepromoter-containing fragment was designed with flanking AccI sites toallow cloning into the compatible, and supposedly, unique BstB I sitelocated in pBHR1 and the derivative pDD47 plasmid. However, digestion ofpDD47, and subsequently pBHR1, revealed the presence of two BstBI sites.Evidently, the sequence of pBHR1 as reported in the literature (GenBank:Y14439.1) is not entirely correct. Due to this discrepancy additionalcloning steps were required to add the cloned Pm promoter to pDD47.

As shown in FIG. 8A, pDD47 contains unique EcoRI and AgeI sites thatflank the annotated BstB I site targeted for insertion of the promoter.It also contains an NgoM IV site downstream of the AgeI site. The 763 bpEcoRI-NgoMIV fragment of pDD47 was excised and cloned into EcoRI-NgoMIVcut pREZ6 to generate pDD49 (FIG. 8B). pREZ6, also diagrammed in FIG.8B, is a derivative of pBluescript SK+(Stratagene, LaJolla, Calif.) inwhich a short polylinker sequence (ttaattaagggtttaaactac (SEQ IDNO:142)) was inserted at the unique DraIII site of pBluescript SK+. Inthis construct, the BstBI of interest is unique, so the AccI fragment ofpJ201:11352 which contains the Pin promoter was excised and cloned intothe BstBI site of pDD49 to create pDD50. Subsequently, the EcoRI-AgeIfragment of pDD50 was excised and ligated to the 5055 bp EcoRI-AgeIfragment of pDD47 to create the expression vector pDD54 which is shownin FIG. 8C. pDD54 was used as the expression vector in the initialcloning of K4 capsule genes for transfer into, and expression in,alternative hosts as described below and in Examples 6, 7, 8 and 9.

The three synthetic gene fragments kpsFEDUCS (FS segment), kpsMTAJbABCFG(MG segment) and kfoDIEH (DH segment) were received from the synthesisvendor, DNA2.0 (Carlsbad, Calif.). The synthetic DNAs were provided asfragments cloned in the plasmid vector, pJ241. FIG. 8D shows plasmiddiagrams of these constructs. The synthetic genes were assembled into asingle operon which was subsequently cloned into pDD54. The first stepin this process was to combine the FS and MG segments into a singlefragment. Two plasmids were constructed that combine the FS segment andthe MG segment in different permutations on the plasmid vector, pJ241.

Aliquots of plasmids pJ241:10662 and pJ241:10664 were digested withSbfI+BglII, treated with alkaline phosphatase and gel-purified using theQIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, Calif.) according tothe vendor protocol. In parallel, aliquots of pJ241:10662 andpJ241:10664 DNA were digested with SbfI plus BamHI and the resultingapproximately 9.1 kb and approximately 8.0 kb SbfI-BamHI fragmentscontaining the synthetic MG and FS gene segments, respectively, weregel-purified as above. The gel-purified approximately 9.1 kb SbfI-BamHIMG segment was ligated into the SbfI plus BglII digested andphosphatased pJ241:10664 vector which contains the FS gene segment.Although BamH I and BglII enzymes recognize different sequences, GGATCCvs AGATCT respectively, they produce an identical 4 bp overhang (GATC)and therefore the digestion products can be ligated together butresulting ligation products cannot subsequently be recognized by eitherenzyme. The resulting recombinant plasmid, designated pDD37, is shown inFIG. 8E. This construct retains the SbfI site 5′ to the synthetic genesand the BamHI site, present in pJ241:10664, 3′ to the synthetic genes.The synthetic gene set kpsMTkfoABCFGkpsFEDUCS (MGFS segment), cantherefore be excised as an SbfI-BamHI fragment of approximately 17.1 kb.Similarly, the gel-purified SbfI-BamHI approximately 8.0 kb FS segmentwas ligated into the SbfI and BglII digested and phosphatase treatedpJ241:10662 vector which contains the MG gene segment. The resultingrecombinant plasmid, designated pDD38, is shown in FIG. 8E. Again, thisconstruct retains the SbfI site 5′ to the synthetic genes and the BamHIpresent in pJ241:10662 3′ to the synthetic genes. Therefore, thissynthetic gene set, kpsFEDUCSkpsMTkfoABCFG (the FSMG segment) can beexcised as an SbfI-BamHI fragment of approximately 17.1 kb.

Synthetic genes kfoD, kfoI (or orf3), kfoE and kfoH (or orf1) (the DHsegment), contained in pJ241:10663 (see FIG. 8D), were cloned intoplasmids pDD37 and pDD38. Plasmids pDD37 and pDD38 were digested withEcoRI, treated with alkaline phosphatase and gel-purified using theQIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, Calif.) according tothe vendor protocol. The unique EcoRI site in each of these plasmids islocated in the intergenic region separating kfoC and kfoF. The DHsegment, containing synthetic genes kfoD, kfoI, kfoE and kfoH, wasexcised from pJ241:10663 as a approximately 4.2 kb EcoRI fragment andgel-purified. This fragment was ligated into both of the EcoRI-cut andphosphatased pDD37 and pDD38 plasmids. Resulting recombinants weretested for orientation of the approximately 4.2 kb EcoRI fragment bycutting with diagnostic restriction enzymes. Recombinants that containedthe added DH segment in the correct orientation were readily obtained.The resulting plasmids, pDD51, derived from pDD37, and pDD52 derivedfrom pDD38, are shown in FIG. 8F. These constructs each contain all ofthe K4 capsule cluster genes but, as shown, the gene order differs forthe two plasmids: in pDD51 the gene order is kpsMTkfoABCDIEHFGkpsFEDUCS,in pDD52 the order is kpsFEDUCSkpsMTkfoABCDIEHFG. In both cases theentire K4 gene set can be excised as an SbfI-BamHI fragment ofapproximately 21 kb. The K4 capsule genes from these plasmids weresubcloned into the expression vector pDD54, described above, to generateexpression plasmids pDD57 and pDD58, respectively. Both of theseplasmids are illustrated in FIG. 8G. The entire synthetic K4 capsulegene set was excised from pDD51 and pDD52 as an approximately 21 kbSbfI-BamHI fragment, gel-purified using the QIAEX II Gel Extraction Kit(Qiagen Inc., Valencia, Calif.) according to the vendor protocol andcloned into SbfI-BamHI digested pDD54.

In pDD57 and pDD58 the entire K4 capsule gene set (17 genes) is undercontrol of the Pm promoter and the XylS regulatory protein encoded bythe xylS gene. The pDDS4 and pDD58 plasmids were originally constructedin the E. coli “TOP10” strain, a commercially available (Invitrogen,Carlsbad, Calif.) strain that contains a number of mutations thatenhance its utility for gene cloning. The plasmids were alsosubsequently transferred into another E. coli host (“DH5α”) that iscommonly used in recombinant DNA experiments. These E. coli strains arenot ideal candidates for development as production platforms. Therefore,in initial experiments, pDD54 and pDD58 were transformed into moresuitable E. coli K-12 strains and the resulting strains tested forchondroitin production as described below in Example 6.

Additional expression plasmids also were constructed by modification ofpDD57 and pDD58. A tetracycline-resistance gene was added to expressionplasmids pDD57 and pDD58 as detailed below. Tetracycline-resistancepotentially has two advantages as a selection for plasmid introductionand maintenance. First, tetracycline-resistance (TcR) is typically amore stringent selection for plasmid maintenance because the resistancemechanism is based on transport of the antibiotic out of the cell, noton inactivation of the antibiotic, as is the case for chloramphenicoland many other antibiotics. Therefore, the effective concentration ofselective agent in the culture medium is unaltered by cell growth andmetabolism. Second, spontaneous chromosomal mutations conferringresistance to tetracycline are uncommon, and were not observed in A.campestris. In contrast, spontaneous chromosomal mutations conferringresistance to chloramphenicol were observed in X. campestris in plasmidtransfer experiments such as those described in Example 6. Thesemutations can potentially obscure CmR transformants/exconjugants thatacquired a plasmid of interest, such as pDD57 or pDD58.

Expression plasmids pDD57 and pDD58 were modified by addition of a genethat confers the property of tetracycline-resistance (TcR) while thechloramphenicol-resistance (CmR) property of these plasmids wasretained. A tetracycline-resistance gene (tetR) present in plasmidpCX027 (described in Example 3 and FIG. 7B above), and derived from theE. coli plasmid vector pBR322, was amplified by PCR and cloned into theunique BamHI sites present in pDD57 and pDD58. In the process ofamplifying and cloning the tetR gene, this gene was modified as follows.PCR primers added a BglII site at the 5′ end of the tetR gene, upstreamof the promoter, and a BamHI site 3′ to the tetR stop codon. Primersfurther modified the gene to eliminate an internal BamHI site (withoutchanging the amino acid sequence of the protein) and to eliminate theso-called “anti-let” promoter normally present on the fragment that wasamplified. This promoter is located near the tetR promoter but directstranscription in the opposite direction (Balbis et al., Gene 1986;50:3-40). This modified tetR gene was created by performing two PCRreactions that amplified two overlapping segments of the tetR gene andintroduced the desired sequence changes. Subsequently a these twofragments were joined together by a subsequent PCR splicing reaction togenerate the tetR gene and promoter region of the desired sequencehaving the BglII site at the 5′ end of the tetR gene, upstream of thepromoter, and a BamHI site 3′ to the tetR translational stop codion.

The first PCR reaction (Reaction A) employed primers DHD218 (SEQ IDNO:113) and DHD219 (SEQ ID NO:114) to amplify approximately 400 bp ofDNA including the amino-terminal portion of the retR coding sequence andupstream promoter sequence. The second reaction (Reaction B) employedprimers DHD220 (SEQ ID NO:115) and DHD221 (SEQ ID NO:116) to amplifyapproximately 900 bp of DNA including the remainder of tetR codingsequence and translational stop codon. Sequences of these primers are asfollows shown below. The shaded sequence shown in DHD218 replaces thesequence ATCGATAAGCTT (nucleic acids 2843-2854 of SEQ ID NO:141) that ispresent in pCX027 and, in so doing, eliminates the ClaI and HindIIIsites located in the tetR promoter region and changes the sequence ofthe −10 region of the anti-tet promoter. The complementary shadedsequences of DHD219 and DHD220 create a silent mutation that eliminatesthe BamHI site of the tetR gene of pCX027. This mutation changes a CTCleucine codon to a TTG leucine codon and thus does not alter the aminoacid sequence of the TetR protein.

DHD218 (SEQ ID NO: 113)

GGTAGTTTATCAC >3  DHD219 (SEQ ID NO: 114)

DHD220 (SEQ ID NO: 115)

DHD221 (SEQ ID NO: 116) 5> GCGGATCCTTCCATTCAGGTCGAGGTG >3

PCR Reactions A and B were performed using PfuUltra II polymerase(Stratagene, LaJolla, Calif.). In each 40 μL reaction, Pfu reactionbuffer (supplied by the vendor) was added to a final concentration of1×, primers were added to a final concentration of 0.4 μM each, dNTPswere added at a final concentration of 200 μM each, 1 ng of pCX027plasmid DNA was added as template and 2.5 units of PfuUltra IIpolymerase were added. PCR Reactions A and B were performed in aRoboCycler® Gradient 96 thermocycler (Stratagene, LaJolla, Calif.) usingthe following cycling parameters: 1 cycle of 1 min. at 95° C.: 30 cyclesof 30 sec. at 95° C., 30 sec. at 55° C., and 30 sec. at 72° C.; 1 cycleof 5 min. at 72° C.; and a hold at 6° C. The products of these reactionswere purified using the Qiagen QIAquick PCR Purification Kit (QIAGEN,Valencia, Calif.) according to the vendor protocol, and analyzed byagarose gel electrophoresis. The sizes of PCR products observed wereconsistent with the expected sizes for the products of both Reaction A(395 bp) and Reaction B (920 bp). These fragments were excised from thegel and eluted from the gel slices using the QIAquick Gel Extraction Kit(QIAGEN Inc., Valencia, Calif.) according to the vendor protocol andrecovered in 30 μL of EB elution buffer. The gel-purified fragmentsserved as templates in the subsequent PCR splicing reaction; ReactionSP. In a 50 μL reaction, Pfu reaction buffer was added to a finalconcentration of 1×, primers were added to a final concentration of 0.4μM each, dNTPs were added at a final concentration of 200 μM each, 3 μLof each of the gel-purified reaction products of Reactions A and B wereadded as template and 2.5 units of PfuUltra 11 polymerase were added.PCR Reaction SP was performed in a RoboCycler® Gradient 96 thermocycler(Stratagene, LaJolla, Calif.) using the following cycling parameters: 1cycle of 1 min. at 95° C.; 30 cycles of 30 sec. at 95° C., 30 sec. at55° C., and 30 sec. at 72° C.; 1 cycle of 5 min. at 72° C.; and a holdat 6° C. The product of this reaction was purified using the QIAGENQIAquick PCR Purification Kit (QIAGEN, Valencia, Calif.) according tothe vendor protocol, and was analyzed by agarose gel electrophoresis. Astrong band was observed at a position consistent with the expected sizeof the product of the PCR splicing reaction, 1295 bp.

This PCR product was digested with BglII and BamHI and ligated withBamHI-digested pDD57 and pDD58. Ligation products were used to transformE. coli TOP10 (Invitrogen, Carlsbad, Calif.) and transformants that hadacquired tetracycline resistance were selected by plating at 30° C. onLB plates containing 10 μg/mL tetracycline. Resultingtetracycline-resistant transformants were screened by diagnostic PCRreactions and restriction digests to confirm the presence, and determinethe orientation, of the tetR gene. Plasmids having the desired structurewere identified and designated pDD61 (pDD57::tetR) (SEQ ID NO:143) andpDD62 (pDD58::tetR) (SEQ ID NO:144). Diagrams of these plasmids areshown in FIG. 8H. The analogous insertion of the tetR gene was made intothe BamHI site of vector pDD54 to generate pDD63; shown in FIG. 8I. Thisplasmid can serve as a TcR vector-only control for experiments with anyof the TcR plasmids that express cloned K4 genes.

The synthetic gene set contains restriction sites that allow non-polardeletions of any gene(s) of interest to be created. The set of fourgenes kfoDIEH was deleted by deletion of a single 4.2 kb EcoRI fragment.This 4.2 kb EcoRI fragment was deleted from expression plasmids pDD57and pDD58 and from their respective TcR derivatives, pDD61 and pDD62which are described above. These four plasmids, shown in FIGS. 8O and8H, all contain 3 EcoRI sites. Two sites define the 4.2 kb fragment ofinterest and the third site cleaves within the coding sequence of theplasmid gene that confers chloramphenicol-resistance (CmR). Each ofthese plasmids was digested to completion with EcoRI and the resultingdigestion products were religated. Following transformation with theligation products, CmR transformants were selected and analyzed byrestriction endonuclease digestion. Plasmids deleted for the 4.2 kbEcoRI fragment were readily obtained in all instances. Plasmids pDD59,pDD60, pDD67 and pDD66 are the 4.2 kb EcoRI fragment deletionderivatives of pDD57, pDD58, pDD61 and pDD62, respectively, and all aredeleted for the kfoDIEH genes. These plasmids are depicted in FIG. 8J.

Western blot analyses of expression of the cloned K4 genes (See Example5 below) indicated that in E. coli strains containing pDD66, theexpression of the kpsFEDUCS genes was less than optimal. Therefore,pDD66 was modified to incorporate a promoter (Pm) in the intragenicregion between kfoG and kpsF. In pDD66, this intergenic region containsa unique PacI site and two ClaI sites as shown in FIG. 8K. Digestionwith PacI and ClaI, excises 2 fragments, a 34 bp C/al fragment and a 12bp ClaI-PacI fragment and leaves the larger vector fragment with ClaIand PacI ends. A 127 bp PacI-ClaI DNA fragment having the sequence:

(SEQ ID NO: 80) TTAATTAATGTTTCTGTTGCATAAAGCCTAAGGGGTAGGCCTTTCTAGAGATAGCCATTTTTTGCACTCCTGTATCCGCTTCTTGCAAGGCTGGACTTATCCCTATCAAACCGGACACTGCATCGAT,was inserted into the ClaI-PacI digested pDD66 vector fragment togenerate pBR1052. The added 127 bp PacI-ClaI fragment includes a copy ofthe Pm promoter sequence. As shown in FIG. 8K, in pBR1052 the added copyof the Pm promoter is oriented so that transcription initiation at thispromoter can result in RNA transcripts that include the kpsFEDUCS genes.

Expression plasmids, pDD66 and pBR1052 have been described above. Toconstruct gene replacement vectors in order to insert the K4 chondroitinbiosynthetic genes into the chromosome (as described in Example 10below), the K4 chondroitin biosynthesis genes from pDD66 and pBR1052were cloned into the pMAK-CL replacement vector which is described abovein Example 3. The pMAK-CL vector, diagramed in FIG. 8L, contains clonedDNA regions upstream and downstream of the colanic acid (CA) genecluster and a unique AscI cloning site at the junction of these regions.As detailed in Example 3, this vector was used to construct a deletionof the entire CA gene cluster in E. coli K-12 W3110 to generate strainMSC188. The K4 gene expression cassettes were excised and gel-purified,using the QIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, Calif.)according to the vendor protocol, from pDD66 and pBR1052 asapproximately 19 kb AscI fragments and these fragments were ligated withpMAK-CL DNA that was AscI-digested, phosphatase-treated, andgel-purified. Transformants were selected for resistance totetracycline. The gene conferring resistance to tetracycline is presenton the AscI fragments of pDD66 and pBR1052, along with the Pm promoterand the upstream and downstream transcription terminator sequences.Derivatives of pMAK-CL that contained the AscI fragments of pBR1052 orpDD66 were identified and designated pDD74 and pDD76 respectively. Theseplasmids are diagramed in FIG. 8L.

DNA regions upstream (5′) and downstream of (3′) the E. coli fhuA genewere cloned by PCR, assembled and sequenced, and this deletion fragmentmoved into the pMAK705 suicide plasmid to create a replacement vectorfor the fhuA locus termed pMAK705-ΔfhuA, or pDD73 (FIG. 8M). DNAsegments upstream and downstream ofthe fhuA gene were amplified by PCRfrom genomic DNA prepared from E. coli K-12 strain W3110 (See Example 3)and these two fragments were subsequently joined together by asubsequent PCR splicing reaction. This procedure facilitated theaddition of a PstI site at the junction of the upstream and downstreamDNA segments.

In the initial round of PCR one reaction (Reaction A) employed primersDHD236 (SEQ ID NO:108) and DHD237-S(SEQ ID NO:109) to amplifyapproximately 800 bp of DNA upstream of the fhuA gene and a secondreaction (Reaction B) employed primers DHD238-S (SEQ ID NO:110) andDHD239 (SEQ ID NO:11) to amplify approximately 950 bp of DNA downstreamof the fhuA gene. Sequences of these primers are as follows:

DHD236 (SEQ ID NO: 108) 5>CGCAAGCTTCGTACCGAAAGATCAGTTGC>3  DHD237-S(SEQ ID NO: 109) 5>CCAAAAGAGAAATCTGCAGTAGATGGGATGTTATTTTACCG>3  DHD238-S(SEQ ID NO: 110) 5>ACATCCCATCTACTGCAGATTTCTCTTTTGGGGCACGG>3  DHA239(SEQ ID NO: 111) 5>GCTCTAGACATCTGCCATAACAACGGAG>3 

PCR Reaction A was performed using PfuUltra IT polymerase (Stratagene,LaJolla, Calif.). In a 50 μL reaction, Pfu reaction buffer (supplied bythe vendor) was added to a final concentration of 1×, primers were addedto a final concentration of 0.4 μM each, dNTPs were added at a finalconcentration of 200 μM each, 50 ng of W3110 genomic DNA was added astemplate and 2.5 units of PfuUltra 11 polymerase were added. PCRReaction A was performed in a RoboCycler® Gradient % thermocycler(Stratagene, LaJolla, Calif.) using the following cycling parameters: 1cycle of 1 min. at 95° C.; 30 cycles of 1 min. at 95° C., 1 min. at 55°C., and 1 min. at 72° C.; 1 cycle of 4 min. at 72° C.; and a hold at 6°C.

PCR Reaction B was performed using Herculase polymerase (Stratagene,LaJolla, Calif.). In a 50 μL reaction, Herculase reaction buffer(supplied by the vendor) was added to a final concentration of 1×,primers were added to a final concentration of 0.4 μM each, dNTPs wereadded at a final concentration of 200 μM each, 25 ng of W3110 genomicDNA was added as template and 2.5 units of Herculase polymerase wereadded. PCR Reaction B was performed in a RoboCycler® Gradient 96thermocycler (Stratagene, LaJolla, Calif.) using the following cyclingparameters: 1 cycle of 2 min. at 92° C.: 33 cycles of 30 sec. at 95° C.,30 sec. at 50° C., and 1 min. at 72° C.; 1 cycle of 10 min. at 68° C.;and a hold at 6° C.

The products of these reactions were purified using the QIAGEN QIAquickPCR Purification Kit (QIAGEN, Valencia, Calif.) according to the vendorprotocol, and analyzed by agarose gel electrophoresis. The sizes of PCRproducts observed were consistent with the expected sizes for theproducts of both Reaction A (832 bp) and Reaction B (949 bp). Thesefragments were excised from the gel and eluted from the gel slices usingthe QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.)according to the vendor protocol and recovered in 30 μL of EB elutionbuffer. The gel-purified fragments served as templates in the subsequentPCR splicing reaction; Reaction SP. In a 50 μL reaction, Pfu reactionbuffer was added to a final concentration of 1×, primers were added to afinal concentration of 0.4 μM each, dNTPs were added at a finalconcentration of 200 μM each, 3 μL of each of the gel-purified reactionproducts of Reactions A and B were added as template and 2.5 units ofPfuUltra II polymerase were added. PCR Reaction SP was performed in aRoboCycler® Gradient 96 thermocycler (Stratagene, LaJolla, Calif.) usingthe following cycling parameters: 1 cycle of 1 min. at 95° C.; 33 cyclesof 30 sec. at 95° C., 30 sec. at 60° C., and 40 sec. at 72° C.; 1 cycleof 5 min. at 72° C.; and a hold at 6° C. The product of this reactionwas analyzed by agarose gel electrophoresis. A strong band was observedat a position consistent with the expected size of the product of thePCR splicing reaction, 1750 bp. This band was excised from the gel usingthe QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.)according to the vendor protocol. This fragment was then cloned into thepCR-Blunt II-TOPO cloning vector (Invitrogen, Carlsbad, Calif.)according to the vendor protocol and the sequence of the cloned PstIfragment was determined (SEQ ID NO: 112).

This sequence matched the expected sequence for the DNA segmentsupstream and downstream of the fhuA gene based on the reported genomesequence for W3110 (GenBank, AP009048) and showed the addition, at thejunction of the upstream and downstream segments, of the 6 bp PstI sitederived from primers DHD237-S and DHD238-S. It also confirmed theaddition of a HindIII site at the 5′ end of the upstream DNA segment andan XbaI site at the 3′ end of the downstream DNA segment which derivefrom primers DHD236 and DHD239 respectively. The sequence-verified PCRfragment was excised from the pCR-Blunt II-TOPO vector and gel-purifiedas a HindIII-XbaI fragment of 1739 bp and ligated with thetemperature-sensitive pMAK705 vector (See Example 3) which was digestedwith HindIII and AbaI and treated with Antarctic Phosphatase (NewEngland BioLabs, Ipswich, Mass.) according to the vendor protocol.Ligation products were used to transform E. coli NEB5α (New EnglandBioLabs, Ipswich, Mass.) and chloramphenicol-resistant transformantsobtained from plating at 30° C., the permissive temperature for pMAK705replication, were analyzed by digestion with PstI, and XbaI plusHindIII, to identify those recombinants carrying the 1739 XbaI-HindIIIbp fragment containing the cloned DNA regions upstream and downstream ofthe E. coli fhuA gene. One such recombinant plasmid was designated aspDD73 (FIG. 8M) and was used in subsequent experiments.

The xylS regulatory gene was cloned into the pDD73 replacement vector asfollows. The xylS gene was excised from pDD42 as a PstI fragment andcloned into the PstI site of pDD73 to generate pDD77, which is diagramedin FIG. 8N. The PstI fragment of pDD77 that contains the xylS gene isidentical to the xylS-containing PstI fragment present in expressionplasmids pDD66 and pBR1052, the parent vector pDD54 and pDD63 thetetracycline resistant derivative of pDD54.

As detailed in Example 10 below, a synthetic optimized version of thexylS gene promoter, ribosome binding site, and 5′ untranslated region(UTR) was designed and synthesized, and those modified sequences wereintroduced into the xylS replacement vector pDD77 and subsequently intothe chromosome. A 257 bp BlpI-BglII fragment (SEQ ID NO:140) wassynthesized by a commercial vendor (DNA2.0) and the synthetic DNAcontaining the modified sequences was cloned into the xylS replacementvector, pDD77, as a Blp I-Bgl II fragment in place of the native BlpI-Bgl II fragment containing the native xylS regulatory sequences. Theplasmid containing the modified xylS was termed pDD79 (FIG. 8N).

A replacement vector was constructed in order to insert a copy of thekfoABCFG gene segment into the E. coli K-12 chromosome at the fhuAlocus. The kfoABCFG gene segment (without the Pm promoter) was excisedfrom pCX039 on a PsIt fragment and this fragment was cloned into thecompatible NsiI site of pDD79 which is unique in this plasmid. In theresulting plasmid, pDD80 (FIG. 8O), the kfoABCFG genes are transcribedby the synthetic xylS promoter, which was designed to be a strongconstitutive promoter.

Deletion derivatives of the pDD66 and pDD67 expression plasmids wereconstructed in order to evaluate the roles of individual genes or groupsof genes. Construction of these derivatives utilized the flankingrestriction enzyme sites designed into the synthetic K4 gene fragmentsas described above. The kpsC gene (K4 region 1) was deleted from pDD66by digestion of 0.6 μg DNA with 10 U SacI for 2 hours in a 10 μLreaction followed by heat treatment of the reaction (to inactivate theenzyme) and ligation (with 1 mM ATP plus T4 DNA ligase) in a 12 μLreaction. Half of this reaction was transformed into E. coli DH5α(Invitrogen) with plating to LB Tc5 at 30° C. In pDD66, the kpsC gene isflanked by SacI sites, but there is also a third SacI site in the vectorsuch that digestion results in a third fragment containing the tetR genebut not the plasmid origin of replication. Therefore, TcR transformantswere expected to contain plasmids comprised of at least thevector/origin fragment plus the tetR fragment. Transformants werescreened for plasmids containing these two SacI fragments but lackingthe kpsC SacI fragment, and candidate pDD66ΔkpsC clones were furtherscreened by SalI digestion for those with the desired orientation of theformer two SacI fragments. One such plasmid was named pCX045 (FIG. 8P).

The kpsT gene (K4 region 3) in pDD66 is flanked by MluI restrictionsites and there are no other MluI sites in the plasmid. Using stepssimilar to those described above, pDD66 was digested with MluI followedby re-ligation to generate a pDD66ΔkpsT derivative that was namedpCX048. (FIG. 8P)

Plasmid pCX039 was created from pDD67 (described above, see FIG. 8J) bydeletion of the K4 regions 1 and 3 genes. Plasmid pDD67 (1.5 μg) wasdigested simultaneously with enzymes PmlI and Mu (10 U each), followedby treatment with T4 DNA polymerase (1.5 U) plus dNTPs (150 μM each) at12° C. for 15 min. to fill in the overhangs (leaving blunt ends)generated by MluI. PmlI digestion leaves blunt ends. Treated pDD67 wassubsequently incubated with T4 DNA ligase and transformed into E. coliTOP10 (Invitrogen) followed by selection for Tet-resistance andscreening for Cm-resistance. Colony PCR on 48 double antibioticresistant colonies was performed with primers DHD229 and DHD231.

DHD229 (SEQ ID NO: 81) AAGGCGACAAGGTGCTGATG DHD231 (SEQ ID NO: 82)CAATGCGACGGATGCTTTCG

Fourteen of the 48 isolates yielded PCR products approximating the 678bp expected for the desired construct as determined by agarose gelelectrophoresis. The plasmids in six of eight selected candidates wereof expected size (by agarose gel electrophoresis), and two chosenplasmid isolates contained the desired DNA sequence at the PmlIMluIjunction. One plasmid was named pCX039 (FIG. 8Q). It contains xylS plusthe K4 region 2 genes kfoABCFG driven by the Pm promoter.

The kfoB gene in pCX039 (plus its ribosome binding site) is closelyflanked by BstBI restriction sites, and there is a third BstBI site inthe vector backbone. Therefore, digestion of pCX039 with BstBI resultsin three fragments: the kfoB gene fragment, a large fragmentencompassing the plasmid replication origin, the Tet-resistance gene,and the kfoCFG genes, and a fragment containing the Cm-resistance geneplus Pm/kfoA (see FIG. 8Q). To create a derivative of pCX039 lacking thekfoB gene, plasmid (600 ng) was completely digested with BstBI (10 U)for 90 min. at 65° C. The enzyme was removed from the reaction with theMinElute kit (QIAGEN) with a final elution in 12 μL Elution Buffer.Approximately 250 ng (5 iLL) of this digest was incubated with T4 DNAligase and transformed into E. coli DH5α (Invitrogen) with selection forCm-resistance. By selecting for Cm-resistance, plasmids containing atleast the large vector fragment (i.e., kfoCFG/origin) plus theCm/Pm/kfoA fragment should be obtained. Plasmids in 8 selectedtransformants were analyzed by restriction digests, and 5 were found tobe lacking the kfoB BstBI fragment and to have the other two fragmentsin the desired relative orientation. The plasmid in one such isolate wasnamed pCX044 (FIG. 8Q; xylS plus kfoACFG). One skilled in the art willrecognize that an identical plasmid structure could be obtained bypartial plasmid digestion with BstBI enzyme.

As described above in this Example, plasmids pDD66 and pDD67 contain 13K4 genes in different arrangements: pDD66—Pnm/kpsMT/kfoABCFG/kpsFEDUCS;pDD67—Pm/kpsFEDUCS/kpsMT/kfoABCFG. Most of the K4 genes (together withtheir respective ribosome binding sites) in these plasmids are closelyflanked by pairs of restriction enzyme sites that cut only two or threetimes within the plasmids. These features (and other sequence elementsdescribed above) allow for selective, non-polar deletion of individualK4 genes. Using the steps described above for the creation of pCX044from pCX039, ΔkfoB derivatives of pDD66 and pDD67 were generated, andthese plasmids, depicted in FIG. 8R, were designated pCX040, and pCX042,respectively. The kfoG genes in pDD66 and pDD67 are closely flanked byNheI restriction sites, but in each plasmid there is a third NheI sitein the coding region for the tetracycline resistance gene. Forgeneration of ΔkfoG derivatives of pDD66 and pDD67, an approachanalogous to that for the generation of the ΔkfoB derivatives wasutilized: complete digestion with NheI, ligation, and selection forTet-resistant transformants of E. coli. This approach selects forregeneration of the tetracycline resistance gene along with the plasmidreplication origin. Plasmids in resulting transformants were screenedfor absence of the kfoG NheI fragment, and pCX041 (pDD66 ΔkfoG) andpCX043 (pDD67 ΔkfoG) were identified and are shown in FIG. 8S. Oneskilled in the art will recognize that identical plasmid structurescould be obtained by partial plasmid digestion with BstBI or NheIenzymes.

Example 5 Antibodies that Recognize K4 Capsule Biosynthesis Proteins

Production of Antibodies:

Antibodies directed against 15 of the proteins that are encoded by theK4 chondroitin biosynthetic gene cluster were produced as describedbelow. These antibodies can be used to assess expression of the clonedK4 chondroitin biosynthetic genes in the alternative hosts and in thenative E. coli K4 strains. They can also be used to assess region 1 andregion 3 gene expression in other group 2 capsule producing E. coli andpotentially used with other serogroup K4 E. coli to assess region 2 geneexpression. Antibodies were generated as follows.

PCR primers were designed to amplify a series of polypeptides, orcomplete proteins, on the order of about 20-30 kDa each in size,corresponding to the 17 genes identified in the K4 capsule gene cluster.The initial set of PCR primers were based on the sequence of the U1-41K4 capsule gene cluster as determined in Example 1. In some instances,cloned PCR fragments were sequence-verified and then subcloned intopQE30 (Qiagen, Inc., Valencia, Calif.), an E. coli plasmid vector, forhigh level expression in E. coli. Alternatively, PCR fragments werecloned directly into the expression vector and then sequenced. The pQE30vector employs a strong bacteriophage T5 promoter under control of theLacI repressor protein to achieve high levels of IPTG inducibleexpression in E. coli. The vector is designed to fuse a poly-His tag atthe amino-terminus of the cloned polypeptide in order to facilitatepurification. Initially, antigens derived from KpsM. KpsF, KpsE. KpsS,KfoC, KfoH and KfoC were expressed in the pQE30 vector as His-taggedpolypeptides and antigens derived from KpsD, KpsU, KpsC, KfoD, KfoI,KfoE and KfoF were expressed without the His-tag. The constructs lackingthe His tag were created by cloning into a derivative of pQE30, termedpQE30-dH, in which the sequence encoding the His residues was deleted.Subsequent expression experiments indicated that the His tag wasrequired for efficient expression of the polypeptide antigens derivedfrom KfoC, KfoH and KfoG, but that other antigens were efficientlyexpressed in pQE30-dH as non-tagged forms. Therefore, most antigens wereexpressed in the non-tagged forms in order to avoid the possibility ofobtaining antisera that recognized an epitope present on the injectedantigen but not present in the native target protein.

For expression of antigens, cultures containing antigen sequences clonedinto pQE30 or pQE30-dH typically were grown at 37° C. in Luria Broth tomid-log phase, and then induced by the addition of 1 mM IPTG. Typically,4 hours post-induction, cells were harvested and fractionated intosoluble and insoluble fractions using BugBuster® Protein ExtractionReagent (Novagen, Madison, Wis.). a detergent-based lysis system,according to the vendor protocols. Typically, over-expression in the T5promoter system leads to accumulation of the expressed polypeptide in aninsoluble form within the E. coli cytoplasm although some expressedpolypeptides are accumulated in a soluble form. The KpsU-derived antigenwas expressed in a soluble form; all other antigens were found topartition to the insoluble fraction. Often, the recombinant protein isthe predominant polypeptide in the insoluble fraction generated by theBugBuster® lysis and extraction procedure. For immunization purposes,expression of these polypeptide antigens was performed in 100 mlshake-flask cultures. Following lysis of induced cultures, insolublefractions of all cultures (except the KpsU antigen-expressing culture)were run on preparative gels. In the case of KpsU antigen, thepolypeptide antigen partitioned to the soluble fraction of the celllysate and therefore that soluble fraction was run on preparative gels.The gel regions containing the protein of interest were excised and sentto the commercial vendor, Open Biosystems (Huntsville, Ala.) for furtherprocessing and subsequent immunization of rabbits to produce antisera.

In the initial experiments, 12 antigens were deemed to be sufficientlywell expressed to warrant purification of the antigen. These 12 antigenswere derived from KpsE, KpsD, KpsU, KpsC, KpsS, KpsT, KfoA, KfoB, KfoI(Orf3), KfoE, KfoH (Orf1) and KfoF. Antigens derived from the proteinsequences of KpsF, KpsM, KfoC, KfoD, and KfoG were not expressed or werepoorly expressed. The polypeptide sequences of poorly expressed antigenswere analyzed with respect to codon usage, and physical properties suchas hydrophobicity and calculated pI. Comparison to these same propertiesas determined for the well-expressed polypeptide antigens did not revealany clear correlations. Codon usage was unfavorable in some poorlyexpressed antigens, such as KfoG, but it was also unfavorable in otherwell-expressed antigens. The KpsM antigen was extremely hydrophobic andthat could potentially affect stability of the expressed antigen, butbecause KpsM is an integral membrane protein, its entire sequence isvery hydrophobic and any polypeptide of significant size derived fromKpsM will be highly hydrophobic.

Additional antigen coding sequences were derived from the synthetic,codon-optimized, genes for KpsM, KpsF, KfoC, and KfoG using thesynthetic K4 gene set described above in Example 2. PCR products derivedfrom the synthetic DNA template were cloned into pQE-30, the 6X-His tagvector, and tested for expression. Synthetic sequence KfoC, KfoG, andKpsF antigens were found to have high, or moderately high, accumulationwhen expressed in the pQE-30 vector with a 6X-His tag. These antigenswere gel-purified from induced cultures as described above and sent toOpen Biosystems (Huntsville, Ala.) for antisera production in rabbits.Synthetic sequence KpsM antigen with a 6X-His tag was not expressed atdetectable levels as determined by Coomassie staining of inducedcultures.

Antisera from immune rabbits were tested by western blot for titer andfor specificity using cell extracts from induced antigen-expressing E.coli strains. All antisera recognized their respective antigens in thesewestern blots. Titers for use were typically 1:1500 with acceptablenon-specific background. Examples of results from western blotsperformed using these antisera are shown in FIG. 9.

As shown in FIG. 9, some antisera (e.g. anti-KfoA, anti-KpsD andanti-KpsS) identified the target protein band in strains carrying thecloned K4 genes with little or no observable non-specific reactivity toother E. coli proteins. In other antisera (e.g. anti-KpsC and anti-KpsF)more non-specific binding was observed, but the target proteins could beclearly identified by comparison to E. coli control strains lacking thecloned K4 genes. Most antisera identified single protein bands as theirspecific targets in western blots, but in some instances (e.g. KfoC)multiple bands were specifically recognized. The KfoC polypeptideappeared to undergo some proteolyic breakdown or processing eitherintracellularly or during processing of extracts prior to the westernblot, and a doublet band, as indicated in FIG. 9. was consistentlyobserved.

Thus, antisera that could detect KpsF, KpsE, KpsD, KpsU, KpsC, KpsS,KpsT, KfoA, KfoB, KfoC, KfoI (Orf3), KfoE, KfoH (Orf1), KfoF and KfoG inE. coli K4 strains, and in recombinant strains expressing the cloned K4capsule gene cluster, and in native E. coli strains that might containsome or all of these genes were successfully raised.

The amino acid sequence of the recombinantly-expressed polypeptide thatwas used to immunize rabbits to generate antisera that recognized theindicated proteins is given below. The antigens that were expressed inthe pQE30-dH vector contain an added MGS sequence at the amino-terminusof the expressed polypeptide which is derived from the plasmidexpression vector and is not present in the sequence of the targetprotein. Antigens that were expressed in the pQE30 vector by cloninginto the BanmH site contain an added MRGSHHHHHHGS (amino acids 1-12 ofSEQ ID NO: 85) sequence at the amino-terminus which is derived from theplasmid expression vector and is not present in the target protein.Antigens that were expressed in the pQE30 vector by cloning into theSacI site contain an added MRGSHHHHHHGSACEL (amino acids 1-16 of SEQ IDNO: 93) sequence at the amino-terminus which is derived from the plasmidexpression vector and is not present in the target protein. Theamino-terminal sequences of the polypeptide antigens that are derivedfrom the expression vector DNA sequences are shaded below.

KfoA-derived antigen (SEQ ID: NO: 83):

ILKKHKFDCIMHFAGAKSVAESLIKPIFYYDNNVSGTLQLINCAIKNDVANFIFSSSATVYGESKIMPVTEDCHIGGTLNPYGTSKYISELMIRDIAKKYSDTNFLCLRYFNPTGAHESGMIGESPADIPSNLVPYILQVAMGKLEKLMV FGGDYPTKDGTKfoB-derived antigen (SEQ ID NO: 84):

LSFSNTINHSLEQSVNDFKTAEASITLEKEHQEIMSLSGIDIGTGDIIFKQSETEEYLIFNVLNDYPDCKELYFELQSNANTPLRVLEKENYKPSFIWETFIKQRQITLDIVNGLYQSSKKIILDNELHTSKQLNAYQAILKELSDSKEE LIQYDLIIKNKTIQVQELECKfoC-derived antigen (SEQ ID NO: 85):

WPSDLTLPPLPESTNDYVWAGKRKELDDYPRKQLIIDGLSIVIPTYNRAKILAITLACLCNQKTIYDYEVIVADDGSKENIEEIVREFESLLNIKYVRQKDYGYQLCAVRNLGLRAAKYNYVAILDCDMKLNKfoI (Orf3)-derived antigen (SEQ ID NO: 86):

TYKENIGKINIHTLPVIIDWLNENRVPYDEVIVGKPWCGDEGFYVDDRAIRPSELCNMTLEEISNMLEQEKKCF KfoE-derived antigen (SEQ ID NO: 87):

GLQLLFGDTLFKKIPAGDDLVAISHSDDNYQWSFFYETELRAVSREDNKNVICGYFSFSKPNFFIREMVTGSFDFTAALKKYHDSYSLASIYVSDWLDFGHINTYYKSKVQYTTQRAFNELCITTKSVIKSSSNESKIEAESKWFETIP  KfoH (Orf1)-derived antigen (SEQ ID NO: 88):

ITIFNIDTIRPNFIFTKFEGENECYIEVFRGDGDNWSFVMPSNDVKNEVIATSEKKQISNLCCTGLYHFSTIKNFISAYEHYKNLPQENWDAGELYIAPIYNYLISNGIKVYYTEINKSDVIFCGTPREYENLQGKfoF-derived antigen (SEQ ID NO: 89):

RKFAELLSEGAIKKDIPILLTDSPEAEAIKLFANTYLAMRIAYFNELDTYASVHGLDTKQIIEGVSLDPRIGQHYNNPSFGYGGYCLPKDTKQLLANYRDVPQNLIQAIVDANTTRKDFVAEDILSRKPKVVGIYRLIMKAGSDNKfoG-derived antigen (SEQ ID NO: 90):

AKGEFVTCHDSDDWSHPEKLFRQISPLLLNPKLICSISDWVRLQDNGIFYARAVYPLKRLNPSSLLFRRADVEQKAGVWDCVKTGADSEFIARLKLIFGDSTVHRIKLPLTLGSHRTDSLMNSPTTGYTSQGISPDRQKYWDSWSRWHIQALRNKESLYIGNSDFTNKNRPFSAPDSILVDTNAIKTALQSAHVNFTKpsT-derived antigen (SEQ ID NO: 91):

LRMIGGIDRPDSGKIITNKTISWPVGLAGGFQGSLTGRENVKFVARLYAKQEELKEKIEFVEEFAELGKYFDMPIKTYSSGMRSRLGFGLSMAFKFDYYIVDEVTAVGDARFKEKCAQLFKERHKESSFLMVSHSLNSLKEFCDVAIVFKDDNAVSFHEDVQEGIEEYITEQNNY KpsF-derived antigen (SEQ ID NO: 92):

HDVPAVQLDASFKTVIQRITSGCQGMVMVEDAEGGLAGIITDGDLRRFMEKEDSLTSATAAQMMTREPLTLPEDTMIIEAEEKMQKHRVSTLLVTNKANK VTGLVRIFDKpsE-derived antigen (SEQ ID NO: 93):

ETEMEKARQRLDASKAELLSYQDNNNVLDPQAQAQAASTLVNTLMGQKIQMEADLRNLLTYLREDAPQVVSARNAIQSLQAQIDEEKSKITAPQGDKLNRMAVDFEEIKSKVEFNTELYKLTLTSIEKTRVEAARKLKKpsD-derived antigen (SEQ ID NO: 94):

FADGDTIIVGPRQHTFSVQGDVFNSYDFEFRESSIPVTEALSWARPKPGATHITIMRKQGLQKRSEYYPISSAPGRMLQNGDTLIVSTDRYAGTIQVRVEGAHSGEHAMVLPYGSTMRAVLEKVRPNSMSQMNAVQLYRPSVAQRQKEMLNLSLQKLEEASLSAQSSTK EEAS KpsU-derived antigen (SEQ ID NO: 95):

WVATDDPRVEQAVQAFGGKAIMTRNDHESGTDRLVEVMHKVEADIYINLQGDEPMIRPRDVETLLQGMRDDPALPVATLCHAISAAEAAEPSTVKVVVNTRQDALYFSRSPIPYPRNAEKARYLKHVGIYAYRRDVLQNYSQLPESMPEQAESLEQLRLMSAGINIRTFEVAATGPGVDTPACLEKVRALMAQELAENAKpsC-derived antigen (SEQ ID NO: 96)

YAGWGLTDDRHPQSALLSARRGSATLEELFAAAYLRYCRYIDPQTGEVSALFTVLQWLQLQRRHLQQRNGYLWVPGLTLWKSAILKPFLQTATNRLSFSRRCTAASAGVVWGVKGEQQWVRAEAQRKSLPLWRMEDGFLRSSGLGSDLLPPLSLVLDKRGIYYDATRPSELEVLLNHSQLTLAHQMRAEKLRQRLVESKL SKYNLGAKpsS-derived antigen (SEQ ID NO: 97):

FITVEEGGVNAYSSLPRDPDFYRKLPDMPTPHVENLKPSTMKRIGHAMWYYLMGWHYRHEFPRYRHHKSFSPWYEARCWVRAYWRKQLYKVTQRKVLPRLMNELDQRYYLAVLQVYNDSQIRNHSNYNDVRDYINEVMYSFSRKAPKESYLVIKHHPMDRGHRLYRPLIKRLSKEYGLDERVIYVHDLPMPELLRHASLI S

Example 6

The synthetic gene set [kpsFEDUCS+kpsMT+kfoABCDIEHFG]producesfructosylated chondroitin when expressed in E. coli K-12.

Plasmids pDD54 and pDD58, described in Example 4 above, were transformedinto MSC188 (E. coli K-12 strain W3110 deleted for the colanic acidbiosynthetic gene cluster as described above in Example 3). Theresulting strains, MSC204 [MSC188 (pDD54)] and MSC206 [MSC188 (pDD58)],were grown in shake flask cultures and tested for chondroitinproduction. Strains were grown overnight from fresh colonies in CYGmedium (20 g/L casamino acids, 5 g/L yeast extract. 2 g/L glucose, pH7.2) plus chloramphenicol (20 μg/mL) at 30° C. and these cultures werediluted to OD A600=0.05 in the same medium. At OD A600 of approximately0.1 (after approximately 1 hour), the inducer m-toluic acid was added toa final concentration of 2 mM. At 4, 8, and 24 hours post-induction, ODA600 values were determined and samples were taken for analysis. CultureODs are given in Table 6-1 below. For each strain at each time point, 10mL samples for polysaccharide analysis were autoclaved (121° C., >15psi, 5 min.) then stored frozen. Two 5 ml aliquots of each strain ateach time point were centrifuged and resulting cell pellets were storedfrozen for subsequent western blot analyses.

As shown in Table 4A. the E. coli K-12 strains, MSC204 and MSC206, grewwell after induction; at 24 hours post-induction the ODs of both ofthese cultures were approximately 7. Culture samples from theseexperiments were assayed using the HPLC-based. chondroitinase-dependentassay for chondroitin and fructosylated chondroitin, as described indetail in Example 14. Culture samples were subjected to thedefructosylation step (acid treatment) prior to enzymatic digestion inthese assays. Culture samples also were assayed by an ELISA assay thatis specific for fructosylated chondroitin (Example 14). Assay resultsare shown in Table 4A.

TABLE 4A Growth and chondroitin production in strains MSC204 and MSC206.ELISA assay HPLC assay^(b) OD chondroitin chondroitin Strain time^(a)A600 μg/mL μg/mL MSC204 4 hr 1.56 0.0   n.d.^(c) MSC204 8 hr 2.58 0.0n.d. MSC204 24 hr 6.65 0.0 0 MSC206 4 hr 1.38 1.2 4.5 MSC206 8 hr 2.571.1 10.1 MSC206 24 hr 7.14 27.9 25.0 ^(a)post-induction^(b)defructosylation step included in assay ^(c)not determined

These results clearly demonstrate that recombinant E. coli K-12 carryingpDD58 (strain MSC206) produces fructosylated chondroitin. The detectionof the polysaccharide by ELISA demonstrates that the recombinantpolysaccharide produced in these strains is fructosylated chondroitinbecause the antiserum used in the ELISA assay is specific for thefructosylated form of chondroitin and does not recognize unfructosylatedchondroitin. The highest level of fructosylated chondroitin productionobserved in this experiment was approximately 25 μg/mL. Fructosylatedchondroitin production was consistently undetectable in the controlstrain MSC204 that carried the vector-only plasmid, pDDS4. There arequantitative differences in fructosylated chondroitin values measuredbetween the ELISA and the HPLC assays for the MSC206 at 4 hr and 8 hrsamples. These differences probably reflect the lower sensitivity of theELISA assay. Typically, the higher the fructosylated chondroitinconcentration in a given sample, the closer is the agreement between theELISA and the HPLC assays.

A subsequent experiment was performed to confirm the production offructosylated chondroitin by MSC206 and to test the effect of inducerconcentration on level of chondroitin produced. A fresh overnightculture of MSC206 was diluted to 0.05 OD A600 and grown at 30° C. in CYGmedium plus chloramphenicol (20 μg/mL) to an OD A600 of approximately0.1. Aliquots of the culture were then induced by addition of m-toluicacid to a final concentration of 0, 0.5, 1.0, or 2.0 mM. Cultures weregrown for 24 hours post-induction at which point ODs were measured andsamples were taken as described above for polysaccharide assays. Also at24 hours post-induction, aliquots of each culture were diluted andplated on LB to quantitate total viable cells and on LB pluschloramphenicol (17 g/mL) to quantitate plasmid-containing viable cells.Growth and chondroitin production of these cultures are summarized inTable 4B.

TABLE 4B Effect of inducer concentration on growth and chondroitinproduction in MSC206. Inducer concentration 0 0.5 mM 1.0 mM 2.0 mM ODA600 at 24 hr 3.08 2.99 2.91 2.29 cfu/mL LB 1.77 × 10⁹ 2.35 × 10⁹ 1.77 ×10⁹ 0.81 × 10⁹ cfu/mL LB + 6.55 × 10⁸ 6.58 × 10⁸ 5.66 × 10⁸ 3.00 × 10⁸Cm¹⁷ % Cm-resistant 27% 28% 32% 37% ELISA assay: 7.4 μg/mL 22.3 μg/mL30.5 μg/mL 12.3 μg/mL chondroitin HPLC assay*: 9.7 μg/mL 25.1 μg/mL 31.1μg/mL 17.5 μg/mL chondroitin *defructosylation step included in assay

As shown in Table 4B, in this experiment, only the highest level ofinducer had a negative effect on growth and viable cell numbers at 24hours. In this experiment, the expression plasmid, pDD58, was not stablymaintained, even though the strain was grown in the presence of theselective antibiotic, chloramphenicol. There appears to be significantloss of plasmid at the 24 hour time point as evidenced by lower titersof colony-forming-units obtained when samples were plated on LB+Cm¹⁷plates as compared to LB plates. However, the fraction ofplasmid-containing cells was not affected significantly by inducerconcentration. Results of the ELISA assay confirm production offructosylated chondroitin in MSC206 and are consistent with the resultsobtained using the HPLC assay when the de-fructosylation step isincluded. The samples with the highest chondroitin titers showed thebest agreement between ELISA and HPLC assays. These results alsodemonstrate the production of fructosylated chondroitin even in theabsence of induction by m-TA addition. However, all induced culturesproduced more fructosylated chondroitin than the uninduced culture, withthe highest level of fructosylated chondroitin being produced by theculture that was induced with 1.0 mM m-TA.

Example 7

Expression of gene set [kpsFEDUCS+kpsMT+kfoABCFG]producesunfructosylated chondroitin when expressed in E. coli K-12 or E. coli B.The kfoB and kfoG genes are not essential for production ofunfructosylated chondroitin, but kfoG is required for optimalproduction.

Prior to this work, the genes that encode proteins responsible forfructosylation of the K4 capsular polysaccharide had not beenidentified. No function had been identified for the proteins encoded bymany of the genes present in region 2 of the K4 capsule gene cluster:kfoB, kfoG, kfoD, kfoE, kfoH (orf1) and kfoI (orf3).

The genes present within region 2 of group 2 E. coli capsules are alltypically involved in synthesis of the polysaccharide or the sugarnucleotide precursors of the polysaccharide (Whitfield 2006). As notedabove (Example 1), the kfoB and kfoG genes encode proteins that arehomologous to those encoded by genes present in capsule clusters ofbacteria known to produce other glycosaminoglycan capsules. Thiscircumstantial evidence suggests a potential role for kfoB and kfoG inbiosynthesis of glycosamninoglycan capsules. In contrast, prior to thepresent invention, no evidence implicated kfoD, kfoI, kfoE and kfoHgenes as being involved in biosynthesis of the chondroitin backbone ofthe K4 capsular polysaccharide. The present inventors hypothesized thatthe kfoD, kfoI. kfoE and kfoH genes might encode proteins that areinvolved in fructosylation of chondroitin although others havehypothesized that the kfoD and kfoE genes were probably not involved infructosylation (Ninomiya et al., 2002 and Krahulec et al., Molec.Biotech., 2005:30:129-134). To test that hypothesis, recombinantplasmids were constructed that did not contain the kfoDIEH gene set butdid contain kpsFEDUCS, kpsMT and kfoABCFG genes. Two such plasmids,termed pDD66 and pDD67, were constructed as described in Example 4above. These two plasmids also contain a gene conferring tetracyclineresistance, so that tetracycline can be used in cell cultures to selectfor plasmid maintenance. A derivative of pDD58, termed pDD62, was alsoconstructed as a control plasmid. The pDD62 plasmid, described in detailin Example 4 above, contains kpsFEDUCS, kpsMT and kfoABCDIEHFG genes andalso contains a gene that confers resistance to tetracycline.

To determine if deletion of the kfoDIEH genes affected biosynthesis ofthe fructosylated chondroitin, pDD62, pDD66 and pDD67 were transformedinto MSC188 or MSC175 (W3110ΔwcaJ described in Example 3 above) and theresulting strains were cultured and assayed for production offructosylated chondroitin and unfructosylated chondroitin. StrainsMSC274 (MSC175+pDD62), MSC279 (MSC188+pDD66) and MSC280 (MSC188+pDD67)were grown in CYG medium in shake flasks at 30° C. with 2 μg/mLtetracycline (Tc) and induced with 1 mM m-TA as indicated. Cultures weresampled at 24 hours post-induction as described above, autoclaved,centrifuged and the resulting supernatants were assayed by the HPLCassay with, or without, a de-fructosylation step.

As shown in Table 5A below, all strains produced chondroitin, but thechondroitin polysaccharide produced by strains containing plasmids pDD66or pDD67 shows no evidence of fructosylation. That is, the chondroitintiters measured by HPLC for MSC279 and MSC280 samples are notsignificantly different for the samples subjected to thede-fructosylation step sample as compared to the samples that were notsubjected to the de-fructosylation step. In contrast, very littlechondroitin is observed when the MSC274 sample is assayed without thede-fructosylation step. Significant chondroitin is only detected in theMSC274 sample that was subjected to the de-fructosylation step. Asdetailed in Example 14, fructosylated chondroitin is not digested by thechondroitinase that is used in the HPLC assay, and is therefore notdetectable by this assay. These data clearly demonstrate that one ormore of the kfoDIEH genes must be required for fructosylation ofchondroitin, but none of these genes is required for chondroitinbiosynthesis. These results again demonstrate that chondroitin isproduced in the absence of induction by m-TA, but that the inducedcultures produced more chondroitin than the uninduced cultures.Surprisingly, the titers of non-fructosylated chondroitin produced byboth MSC279 and MSC280 are greater (2.5- to 4-fold), than titer offructosylated chondroitin produced by MSC274. This result suggests thatthe fructosylation event reduces the efficiency of chondroitinproduction. This is consistent with the observation that in vitro,fructosylated chondroitin is a poor substrate for the KfoC enzyme(chondroitin polymerase) as compared to unfructosylated chondroitin(Lidholt and Fjelstad, J. Biol. Chem. 1997:272:2682-2687).

TABLE 5A Production of unfructosylated chondroitin in E. coli K-12. ODA600 cfu/mL cfu/mL chondroitin (μg/mL) Strain m-TA at 24 hr LB LB + Tc %Tc^(r) + de-fruc.^(a) no de-fruc.^(b) MSC274 0 2.62 1.16 × 10⁹ 0.81 ×10⁹ 70 4.1 6.1 MSC274 1 mM 2.76 1.39 × 10⁹ 0.95 × 10⁹ 68 15.6 0.2 MSC2790 2.74 1.32 × 10⁹ 1.00 × 10⁹ 76 17.1  n.d.^(c) MSC279 1 mM 2.61 0.90 ×10⁹ 0.86 × 10⁹ 96 37.1 37.7 MSC280 0 2.72 1.43 × 10⁹ 0.97 × 10⁹ 69 15.8n.d. MSC280 1 mM 3.03 1.48 × 10⁹ 1.12 × 10⁹ 76 54.4 59.9 ^(a)subjectedto defructosylation treatment; 80° C. at pH 1.5 for 30 minutes ^(b)notsubjected to defructosylation treatment ^(c)not done

These strains show improved plasmid retention, i.e., retention ofantibiotic resistance, as compared to strain MSC206. See Table 4B inExample 6 above for MSC206 plasmid retention data. This probablyreflects the use of tetracycline vs. chloramphenicol for selection ofthe plasmid. Additional experiments could be performed to optimize theconcentration of tetracycline, or other preferred antibiotic, used inorder to maximize plasmid retention, without impairing cell growth, toachieve maximal chondroitin production.

Plasmids pDD66 and pDD67 were transformed into msc139, E. coli B(ATCC11303), and the resulting strains were tested for chondroitinproduction. A control plasmid, pDD63, was also transformed into MSC139.This plasmid. described in Example 4 above, is a derivative of the pDD54vector to which a tetracycline-resistance gene has been added. Itcontains none of the K4 chondroitin biosynthesis genes. Chondroitinproduction in E. coli B containing pDD63 (MSC314), pDD66 (MSC315) orpDD67 (MSC316) was evaluated in shake flasks.

In this experiment, cultures were grown in TB medium containing 5 μg/mLtetracycline (Tc⁵) at 30° C. As described in Example 8 below, growth inTB medium was found to enhance recombinant production of chondroitin inE. coli as compared to CYG medium, and Tc⁵ was found to be an effectiveconcentration for plasmid maintenance without impairing cell growth. Thecultures were inoculated at 0.05 OD A600 and induced at 0.10-0.13 byaddition of 2 mM m-TA. Following induction, cultures were grown at 30°C. for up to 3 days. Strain MSC315 initially grew slower than the othersand was induced several hours later than MSC314 and MSC316 cultures. At48 hours post-induction (42 hours post-induction in the case of MSC315)samples were taken for viable cell counts in the presence or absence oftetracycline and for chondroitin assays by the HPLC method.

Assay results shown in Table 5B below demonstrate chondroitin productionat significant levels in E. coli B when either pDD66 or pDD67 ispresent. No chondroitin was detected in strain MSC314 which contains thepDD63, the “empty vector” control. Plasmid retention (% Tcr) in thisexperiment was approximately 50% for pDD66 and pDD67 whereas there wasno detectable loss of the control vector pDD63.

TABLE 5B Production of recombinant chondroitin in E. coli B. OD cfu/mL %chondroitin^(a) Strain Plasmid Time P-I A600 (×10⁸) Tc^(r) (μg/mL)MSC314 pDD63 48 h 10.8 7.8 100 0 MSC315 pDD66 42 h 7.72 5.1 49 394MSC316 pDD67 48 h 6.70 4.1 56 280 ^(a)HPLC method withoutde-fructosylation treatment

E. coli B does not produce a capsule but does contain a cryptic group 2capsule gene cluster in which the region 2 genes are disrupted by aninsertion element, and regions 1 and 3 genes appear functional(Andreishcheva and Vann, Gene 2004; 484:113-119). To determine whetherthe E. coli K4 region 2 genes can “complement” the E. coli B region 2defect, a plasmid containing only kfoABCFG genes was constructed. Thisplasmid, pCX039, is described in Example 4. Plasmid pCX039 wastransformed into MSC139, E. coli B (ATCC11303), and chondroitinproduction in the resulting strain, designated MSC317, was evaluated inshake flasks. The strain was grown in TB medium plus 5 μg/mL Tc at 30°C. The culture was inoculated at approximately 0.05 OD A600 and inducedwith 2 mM m-TA at an OD of approximately 0.10. At 48 hourspost-induction, samples were taken for viable cell counts in thepresence or absence of tetracycline and for chondroitin assay by theHPLC method.

When assayed on LB plates, 5.9×10⁹ cfu/mL were obtained and the titer ofcfu obtained from parallel platings on LB plates containing 5 μg/mL Tcwas not significantly different. This indicates that the pCX039 plasmidwas quantitatively retained in this experiment. The HPLC basedchondroitin assay was performed without a de-fructosylation step. Thechondroitin titer measured in this assay was 205 μg/mL. This resultdemonstrates that only region 2 K4 genes kfiABCFG are required toachieve chondroitin biosynthesis in E. coli B. In Example 9 below, theregions 1 and 3 genes in E. coli B are shown to function in concert withthe K4 region 2 genes to result in chondroitin secretion, a findingconsistent with those of Andreishcheva and Vann (2004).

As noted above, KfoB and KfoG homologs are encoded in gene clusters ofother glycosaminoglycan-producing bacteria, but the functions of theseproteins remain unknown. As described in Example 4, the kfoB or kfoGgenes were deleted from pDD66 and pDD67 to generate plasmids pCX040,pCX041, pCX042 and pCX043 which are summarized in Table 5C below. Theseplasmids were transformed into host strain MSC188 and cultures of theresulting strains were tested for chondroitin biosynthesis. Cultureswere grown in TB medium at 30° C., induced with 2 mM m-TA at ODA600=0.2, and sampled at 48 hours post-induction for viable cell countsand chondroitin assays. The results of these assays, shown below,suggested that neither gene is absolutely essential for chondroitinbiosynthesis in recombinant E. coli K-12.

TABLE 5C Effects of deletion of kfoB or kfoG on chondroitin production.OD cfu/mL chondroitin^(a) Strain Plasmid A600 (×10⁹) % Tc^(r) (μg/mL)MSC279 pDD66 15.6 0.91 100 564 MSC322 pCX040 = 12.8 0.87 100 676pDD66ΔkfoB MSC323 pCX041 = 18.4 2.81 100 110 pDD66ΔkfoG MSC280 pDD6716.0 1.77 73 384 MSC324 pCX042 = 15.8 1.66 70 358 pDD67ΔkfoB MSC325pCX043 = 14.9 2.24 85 17 pDD67ΔkfoG ^(a)HPLC assay withoutde-fructosylation step

Based on these results, KfoB protein activity appears to be unnecessaryfor chondroitin production in these strains under these growthconditions. In fact, in this experiment the strain carrying the deletionof kfoB from pDD66 produced approximately 20% more chondroitin than thestrain containing pDD66; see MSC279 vs. MSC322. This difference could besignificant but is also within the range of day-to-day variationobserved for chondroitin production in recombinant E. coli. In a repeatexperiment comparing MSC279 and MSC322, enhanced production ofchondroitin by the kfoB deletion strain was not observed. In the pDD67background, the kfoB deletion appeared to have little or no effect onchondroitin production.

Previous published studies of strain of E. coli K4 that was mutated toinactivate kf (i did not report any effect of the kfoG mutation on thelevel of fructosylated chondroitin produced (Krahulec et al., 2005). Incontrast, our results demonstrate that the KfoG protein, although notabsolutely essential for production of chondroitin, appears to berequired for the optimal levels of production of recombinant chondroitinin E. coli. under these growth conditions of this experiment. Deletionof the kfoG gene severely reduced production of chondroitin by pDD66 andpDD67. In the pDD66 background, the strain deleted for kfoG (MSC323)produced only approximately 20% as much chondroitin as the wild typecontrol strain MSC279. Similarly, in the pDD67 background, the straindeleted for kfoG (MSC325) produced only approximately 5% as muchchondroitin as the wild type control strain MSC280.

Example 8

This example demonstrates recombinant production of chondroitin in avariety of growth media, temperatures, and induction conditions.

A variety of different growth media can support production ofchondroitin by recombinant E. coli strains carrying the E. coli K4chondroitin biosynthesis genes. For optimal production of recombinantchondroitin cultures conditions such as medium composition, temperature,inducer concentration, and duration of culture post-induction need to beoptimized.

Initial studies of recombinant production of chondroitin in E. coli usedCYG growth medium (20 g/L casamino acids, 5 g/L yeast extract, 2 g/Lglucose, pH 7.2). A variety of alternative growth media and cultureconditions can be employed to cultivate recombinant E. coli strainscapable of producing chondroitin and to achieve chondroitin production.

One alternative culture medium that is well known to support the growthof E. coli is TB medium (Sambrook, J., Fritsch, E. F. and Maniatis, T.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). This mediumwas tested and found to also support production of chondroitin byrecombinant E. coli. Further, the effect of prolonged incubation time(up to 72 hours post-induction) on chondroitin production was alsotested. Additionally, the effect on chondroitin production of inductionwith 1 mM vs. 2 mM m-TA was also tested.

Strains MSC279 and MSC280 were grown in shake flasks at 30° C. in TBcontaining tetracycline at 5 μg/mL. Cultures were inoculated at 0.05 ODA600 and induced by addition of 1 mM or 2 mM m-TA at approximately(0.1-0.2) OD A600. At 24 and 48 hours post-induction, cultures weresampled for chondroitin assays and for viable cell counts. Results ofthis experiment are summarized in Table 6A below.

TABLE 6A OD cfu/mL % chondroitin^(b) strain m-TA time^(a) A600 (×10⁹)Tc^(r) (μg/mL) MSC279 1 mM 24 10.1 2.21 81 200 48 14.0 1.47 58 411 2 mM24 10.2 2.13 79 238 48 13.5 0.93 59 436 MSC280 1 mM 24 10.2 2.69 76 22248 15.0 2.19 84 407 2 mM 24 9.9 2.08 90 243 48 14.5 1.27 100 474^(a)hours, post-induction ^(b)HPLC assay without de-fructosylationtreatment

These results demonstrate that TB medium can support significant levelsof chondroitin production. Also, extending the culture timesignificantly increased the chondroitin titer, chondroitin levelsapproximately doubled from 24 to 48 hours post-induction. Finalchondroitin titers of 0.4-0.5 g/L were achieved at 48 hourspost-induction. These data also suggest that the higher inducerconcentration can result in greater productivity although the magnitudeof the effect is not large.

Both CYG and TB are “complex” growth media containing hydrolyzed caseinproducts and autolyzed yeast in which the components of the medium arenot chemically defined. In some circumstances it might be desirable toemploy a minimal or defined growth medium for cell culture. Someexamples of possible defined or minimal media include “2XM9” plusglucose or glycerine and with, or without, supplementation with yeastextract (YE). The basal inorganic salt components of “2XM9” are: 22.6g/L Na₂HPO₄-7H₂O, 6 g/L KHzPO₄, 1 g/L NaCl, 2 mM MgSO₄, 0.2 mM CaCl₂,and 2.0 g/L NH₄CI (pH 7.4). To this formulation a carbon source isadded, and other supplements can be added as indicated. Strains MSC279and MSC280 were cultured in shake flasks at 30° C. in 2XM9 thatcontained 10 g/L glucose or glycerine as carbon sources. The effect ofsupplementation of the glucose containing medium with YE at 1 g/L alsowas tested. For this experiment the overnight cultures that served asthe inocula were grown in LB medium. Cultures were inoculated at 0.05 ODA600 and induced by addition of 1 mM m-TA at approximately 0.1-0.2 ODA600. Cultures containing glucose as the carbon source were sampled at24 and 48 hours post-induction for viable cell counts and forchondroitin assays. However, the glycerine cultures grew relativelyslowly at first and at 24 hours post-induction there was only weakgrowth, so growth of the glycerine cultures was extended to 72 hours andsamples were taken at 48 and 72 hours. Table 6B below summarizes theresults of this experiment.

TABLE 6B Growth OD cfu/mL % chondroitin* Strain medium time A600 (×10⁹)Tc^(r) (μg/mL) MSC279 2xM9 + 24 5.16 2.53 89 110 glucose 48 5.28 2.76 80181 MSC280 2xM9 + 24 5.45 3.06 74 52 glucose 48 5.54 3.00 74 94 MSC2792xM9 + 24 6.13 2.36 97 150 glucose + YE 48 5.37 4.46 87 194 MSC2802xM9 + 24 6.06 2.73 79 99 glucose + YE 48 5.80 2.92 90 120 MSC279 2xM9 +48 4.62 4.62 89 182 glycerine 72 5.84 1.94 84 280 MSC280 2xM9 + 48 5.482.78 88 143 glycerine 72 6.35 2.89 81 350 *HPLC assay withoutde-fructosylation

Both strains attained final OD A600 of approximately 5-6 in all threemedia compositions and plasmid retention was relatively goodapproximately 75-90%. Chondroitin was produced in substantial amounts inall media tested. Titers at harvest ranged from approximately 100 to 350μg/mL. Addition of yeast extract to 2XM9 plus glucose medium had, atmost, a modest effect on final chondroitin titers. Despite an initialgrowth lag, glycerine cultures grew to final cell densities similar tothose seen with glucose. Final titers of chondroitin were higher (1.5-to 3-fold) in the glycerine cultures as compared to the glucosecultures. These results provide examples of minimal/defined media thatcan support significant levels of chondroitin production. Further mediadevelopment and optimization can be performed using standard approachesthat are well known to one skilled in the art of microbial fermentationprocess development.

Another experiment demonstrates the effects of growth temperature andextended growth time post-induction times on chondroitin accumulation.Cultures of MSC280 were grown in CYG medium plus 2 μg/mL Tc. Flasks wereincubated at 20° C., 25° C., 30° C., and 37° C. Cultures were inoculatedat approximately 0.05 OD A600 and grown at the indicated temperatures toapproximately 0.1-0.2 OD A600 at which point cultures were induced byaddition of 1 mM m-TA where indicated. One control culture at 30° C. wasnot induced. Samples were collected at 24, 48, and 72 hours postinduction for chondroitin assays and viable cell counts. Results fromthe terminal harvest timepoints are shown below in Table 6C. Under theseconditions, chondroitin production was achieved at all temperaturestested but the best chondroitin titers achieved were at 25° C. and 30°C. Chondroitin accumulation increased significantly during the secondand third days of growth at all temperatures tested (data not shown). At37° C., chondroitin production is substantially lower (approximately10-fold) than at 30° C., and the viability of the 37° C. culture waspoor. Further optimization could be achieved by refining the definitionof the preferred temperature range for chondroitin production by testingadditional temperatures in the range 20° C. to 37° C. Similarly, furtheroptimization could be achieved by refining the effect of time of culturepost-induction upon chondroitin titer.

TABLE 6C OD cfu/mL % chondroitin temp. induction time^(a) A600 (×10⁹)Tc^(r) (μg/mL) 20° C. 1 mM m-TA 24 1.42 0.92 90 12 48 2.24 0.59 100 3672 2.36 0.54 93 55 25° C. 1 mM m-TA 24 2.62 1.63 99 45 48 2.98 n.a.^(b)n.a.^(b) 96 72 3.02 0.84 82 123 30° C. 1 mM m-TA 24 2.97 0.97 76 72 483.38 1.35 82 107 72 3.36 0.57 74 119 30° C. not induced 24 2.83 0.66 7325 48 3.73 0.44 68 42 72 2.82 0.45 80 62 37° C. 1 mM m-TA 24 2.44n.a.^(b) n.a.^(b) 10 48 2.22 0.07 n.d.^(c) 13 ^(a)hours post-induction^(b)data not available ^(c)not determined, no colonies obtained on Tetplates

Additional studies to optimize time of incubation by extending theincubation time even further may further increase chondroitinproduction. Similarly, additional concentrations of inducer may betested to identify the optimal concentration for chondroitin production.

Example 9

This example demonstrates that recombinant chondroitin can be secretedto the culture medium in E. coli K-12 and E. coli B. This examplefurther demonstrates that chondroitin can also be producedintracellularly at high levels.

When E. coli K4 is cultured in liquid media, the capsular polysaccharide(K4P), fructosylated chondroitin, is reported to accumulate in acell-free form in the culture medium and as a cell associated form(Manzoni et al., Biotech. Lett. 1996; 18:383-386, Cimini at al. Appl.Mocrohiol. Biotechnol. E-Publication, E-Pub. October 2009). By analogyto other group 2 capsular polysaccharides, such as those produced by E.coli serotype K1 and E. coli serotype K5, the cell associated form isbelieved primarily to be associated with the outer leaflet of the outermembrane of the cell by means of a lipid anchor (Whitfield, 2006). Thenature of the linkage between the polysaccharide and the lipid anchorhas not been defined at a structural level, nor has the identity of thelipid anchor been determined. Recombinant chondroitin produced anddetected as described in Examples 6-8 above is clearly present in theculture medium. Low speed centrifugation (10 minutes at 3500 g) issufficient to remove cells from the culture medium of samples to beassayed for chondroitin, and significant quantities of chondroitin havebeen detected in the cell-free supernatants. However, all samples thatwere assayed for chondroitin in Examples 6-8 above were autoclaved priorto centrifugation in order to kill the bacteria and thus facilitatesample handling. The autoclaving step could potentially disrupt thelinkage of any cell-associated chondroitin and result in suchcell-associated chondroitin being released fnrom the cell. In order todetermine whether recombinant chondroitin is produced in cell-freeand/or cell-associated forms, an experiment was performed that testedthe effect of autoclaving on partitioning of recombinant chondroitininto supernatant and pellet fractions following centrifugation ofsamples from chondroitin-producing cultures.

Strain MSC279 was grown in a shake flask in TB medium 5 μg/mL Tc at 30°C. The culture was inoculated at approximately 0.03 OD A600 and grown toapproximately 0.1-0.2 A600 at which point it was induced by addition of2 mM m-TA. At 48 hours post-induction samples were taken and assayed forchondroitin. One aliquot of this culture was autoclaved prior to beingcentrifuged, and the resulting supernatant and cell pellet fractionswere assayed for chondroitin according to the HPLC method of Example 14.Another aliquot was centrifuged without being autoclaved and theresulting supernatant and cell pellet fractions were assayed forchondroitin according to the HPLC method of Example 14. A cell pelletfrom a non-autoclaved sample was resuspended in THB (50 mM Tris-HClbuffer with 50 mM sodium acetate pH 8.0) and treated directly withchondroitinase ABC (“CHase”) (without cell lysis) and again centrifugedto generate supernatant and pellet fractions for assay. To test forcell-free chondroitin carried over in residual culture medium, anothercell pellet from a non-autoclaved sample was gently washed in THB thenre-centrifuged. The supernatant from the wash (sample #7) and the cellpellet (without lysis) from the wash (sample #8) were assayed forchondroitin as above. Results of this experiment are shown in Table 7Abelow.

TABLE 7A chondroitin Sample sample #/fraction (μg/mL) autoclaved culture1 supernatant 403 medium 2 pellet (lysis assay) 50 non-autoclaved 3supernatant 217 culture medium 4 pellet (lysis assay) 178 Pelletresuspended + 5 supernatant 130 from non- CHase, CFGed 6 pellet (lysisassay) 66 autoclaved washed 1X, 7 wash superantant 8 cultureresuspended + 8 wash pellet 154 medium CHase, CFGed

In the autoclaved sample, only 11% of the total chondroitin was presentin the cell-pellet (sample #2) whereas 45% of the total chondroitin waspresent in the cell pellet in the sample (sample #4) that was notautoclaved prior to centrifugation. This result indicates that asignificant fraction of the chondroitin produced by MSC279 remainscell-associated and that the autoclaving step disrupts the associationof the chondroitin with the cell. The cell-associated chondroitin in thepellet of the non-autoclaved strain was found to be digested by directCHase treatment of resuspended cells without lysis treatment. Thequantity of surface-associated chondroitin calculated based on theamount of disaccharide released was determined to be 130-154 μg/mL(sample #5 and sample #8) in the original culture. This value issomewhat less than the cell-associated chondroitin titer (178 μg/mL)measured by the “cell lysis” technique of Example 14 (sample #4), whichmay reflect internal chondroitin polymer (as measured in sample #2 andsample #6). However, the data from both assay methods are qualitativelyin agreement as indicating that a significantly higher fraction ofchondroitin is cell-associated in the non-autoclaved sample as comparedto the autoclaved sample. The fact that the cell-associated chondroitinwas digested to disaccharide by CHase treatment of the whole-cellsuspension indicates that this fraction of the chondroitin is associatedwith the outer membrane of the cell in such a way that thepolysaccharide resides outside of the cell and in the culture medium.This is consistent with the expected structure of the capsule.

These results demonstrate that a significant fraction (≧50%) of therecombinant chondroitin produced by strain MSC279 is present in theculture medium and in a form that is not cell associated. The bulk ofthe cell-associated chondroitin produced by MSC279 is attached to thecell but is accessible to the surrounding medium as evidenced bydegradation by added CHase. The production of both cell-free andcell-associated forms of K4P by native E. coli K4 has been previouslyreported by Manzoni et al., Biotechnol. Lett. 1996:18(4):383-386 andCimini et al., Appl. Microbiol. Biotechnol. E-Publication, October 2009.The observation of both forms during recombinant production isconsistent with the notion that the synthesis and secretion ofrecombinant chondroitin in MSC279 proceeds by the same pathways thatoperate in the native strain. This suggests that all of the cloned genesintroduced into MSC279 are functioning in the same manner as in E. coliK4 and that the complete pathway for synthesis and export of the capsulepolysaccharide is functioning in the recombinant strain.

In order to produce chondroitin by bacterial fermentation it ispreferable to use large scale fermentations, which generate volumes ofculture media that are too large for autoclaving to be feasible as amethod for releasing cell-associated chondroitin into the culturemedium. Alternative treatments using elevated temperature in combinationwith acid or base treatment could be employed for large scalemanufacturing.

Similar results for secretion of chondroitin into culture medium werealso obtained with recombinant E. coli B strains that producechondroitin. We observed previously that in recombinant E. coli K-12 thekfoB gene was not essential for chondroitin production. To test forsecretion of chondroitin in E. coli B, MSC347 (MSC139 pCX044 i.e.pCX039ΔkfoB) was grown in TB/Tc⁵ medium at 30° C. and induced at OD A600approximately 0.15 with 2 mM m-TA. At 48 hours, broth samples were takenand centrifuged with, and without, autoclaving to generate supernate andcell pellet fractions. Chondroitin assay results of this experiment areshown below in Table 7B. Autoclaving resulted in greater than 90% of thetotal measurable rCH in the supernatant fraction. In non-autoclavedsamples, however, only approximately 30% of the chondroitin was found inthe supernatant. These results are consistent with findings withrecombinant E. coli K-12 strains, as detailed above, which suggest thatthe autoclave step (5 minutes, 121° C., 15 psi) releases essentially allrCH into the medium. These results further show that the regions 1 and 3genes in E. coli B function to secrete chondroitin when the region 2genes are present.

TABLE 7B OD chondroitin strain description A600 treatment sample μg/mLMSC347 MSC139 12.6 autoclaved supernate 362 pCX044 cell pellet 25 pCX044= non- supernate 107 pCX039 autoclaved cell pellet 242 (ΔkfoB)

Secretion of chondroitin into cell culture medium and release ofcell-associated chondroitin into the culture medium provide a method forobtaining chondroitin that is cell free and can be separated from cellsby centrifugation or filtration and subsequently purified. It is alsopossible to produce intracellular chondroitin by genetic manipulation ofthe chondroitin biosynthesis genes. Intracellular production ofchondroitin could be desirable in order to eliminate viscosity of thefermentation resulting from high levels of polysaccharide in the culturemedium. In addition, the intrinsic limits upon chondroitin productionand the biochemistry of chondroitin biosynthesis in E. coli areincompletely understood. It is possible that intracellular productioncould achieve higher levels of chondroitin than secretion. Therefore,recombinant gene sets that allow accumulation of significant levels ofchondroitin in the absence of secretion into the culture medium wereidentified.

There is evidence in the literature that under certain conditions, otherE. coli capsular polysaccharide can be synthesized and accumulateintracellularly. Electron microscopy (EM) results of Brmnner et al. (J.Bact. 1993:175:5984-5992) suggested that there was some intracellularaccumulation of the E. coli serotype K5 capsular polysaccharide(heparosan) by mutants defective in kpsC and kpsS. Similar observationswere reported by Cieslewicz and Vimr, (J. Bact. 1996; 178:3212-3220) forthe polysialic acid capsular polysaccharide of E. coli K1 when mutantsdefective in kpsC, kpsS, kpsE or kpsT were examined by EM. The levels ofintracellular K1 and K5 polysaccharides were not quantitated in thesestudies.

To determine if mutations in region 1 or region 3 genes could blocksecretion of recombinantly produced chondroitin in E. coli K-12,derivatives of plasmid pDD66 that are deleted for kpsC or kpsT genes(pCX045 and pCX048 respectively) were constructed as described inExample 4. These plasmids were transformed into MSC188 and the resultingstrains were tested in parallel with MSC279 (MSC188 containingunmodified pDD66) for the ability to produce and secrete chondroitin tothe culture medium.

Cultures were grown in TB+Tc5 medium at 30° C., induced with 2 mM m-TAat OD A600 of approximately 0.15, and sampled after 48 hours. For eachstrain at the 48 hour time point, chondroitin was assayed in thesupernates and cell pellets from both autoclaved and non-autoclavedsamples. As shown in Table 7C below, the ODs at 48 hours wereequivalent. In non-autoclaved samples from strains MSC356 and MSC359,which are deleted for kpsC and kpsT genes respectively, chondroitin ispredominantly (approximately 85-90%) localized to the cell pellet. Thisis in contrast to the results with MSC279, the wild-type control, inwhich approximately 50% of the chondroitin is localized to the cellpellet and approximately 50% is present in the supernatant.

TABLE 7C OD chondroitin strain plasmid A600 sample (μg/mL) MSC279 pDD6613.5 non-autoclaved 265 supernatant non-autoclaved pellet 260 autoclavedsupernatant 499 autoclaved pellet 54 MSC356 pCX045 = 13.5 non-autoclaved59 DD66ΔkpsC supernatant non-autoclaved pellet 329 autoclavedsupernatant 400 autoclaved pellet 31 MSC359 pCX048 = 13.4 non-autoclaved50 pDD66ΔkpsT supernatant non-autoclaved pellet 388 autoclavedsupernatant 421 autoclaved pellet 105

As described above (Table 7C) in strain MSC279, most of the chondroitinlocalized to the cell pellet in non-autoclaved samples of MSC279cultures is likely covalently attached to a lipid anchor in the outerleaflet of the cell outer membranes. It is likely that autoclavingdisrupts the association of the cell membrane and chondroitin that iscell-associated in the absence of autoclaving, but the effects ofautoclave treatment of the cells are not fully understood. The nature ofthe association between the cell and the chondroitin that is localizedto the cell pellets of the kpsC or kpsT defective strains is notaddressed by these data. In principle, this chondroitin could be presentin the cell cytoplasm, in the periplasmic space, or still attached tothe outer cell membrane. However, the results for these mutant strainsare consistent with the notion that mutations in kpsC and kpsT blocksecretion of chondroitin and result in intracellular accumulation ofchondroitin. Presence of this chondroitin on the cell surface can betested by resuspending the non-autoclaved cell pellet, subjecting theresuspended cells to CHase digestion and measuring the production of thechondroitin specific disaccharide. Alternatively, electron microscopycan be employed to determine the cellular location of cell-associatedchondroitin produced by MSC356 and MSC359.

An additional experiment, detailed below, was designed to confirm therole of autoclaving in the release of chondroitin from the cell withwild type strain MSC279, and to determine whether E. coli K-12containing only the K4 region 2 genes (MSC346; MSC188 pCX039) couldproduce intracellular chondroitin. Strains MSC279 and MSC346 were grownin TBiTc5 medium at 30° C. and induced at OD A600 approximately 0.15with 2 mM m-TA. After 48 hours, duplicate broth samples were taken togenerate supernate and cell pellet fractions, with, and without,autoclaving prior to centrifugation. Chondroitin assay results fromthese samples are shown in Table 7D below. In the non-autoclaved samplesfrom strain MSC279, which contains the complete chondroitin biosyntheticgene set, chondroitin was approximately evenly distributed betweensupernate (55%) and pellet (45%). In contrast, the non-autoclaved pelletfrom strain MSC346, which contains only region 2 genes, includedapproximately 98% of the chondroitin produced by that culture—little wasfound in the supernate. For both strains, autoclaving resulted inpartitioning of the CH predominantly (>90%) into the supernates.

TABLE 7D CH strain description treatment fraction (μg/mL) MSC279 MSC188pDD66 autoclaved supernate 485 cell pellet 38 non-autoclaved supernate230 cell pellet 186 MSC346 MSC188 pCX039 autoclaved supernate 200 cellpellet 13 non-autoclaved supernate 3 cell pellet 151

These results demonstrate that autoclaving (5 min., 121° C., 15 psi)releases almost all cell-associated chondroitin into the medium.Consequently, the chondroitin detected in the non-autoclaved cell pelletof MSC279 could, in principal, be bound to the outer membrane or have anintracellular location. In the absence of autoclaving, very littlechondroitin is found in the supernatant of the strain MSC346 culture.This result is consistent with MSC346, which lacks all region 1 andregion 3 functions, producing only intracellular chondroitin. Althoughlower amounts of chondroitin are produced by MSC346, as compared toMSC279, the quantity of chondroitin produced is still significant anddemonstrates that chondroitin can be successfully produced using onlycloned genes kfoABCFG in recombinant E. coli K-12. These results alsodemonstrate that this chondroitin can be liberated from the cell byautoclaving and obtained in the supernatant of autoclaved culturesfollowing centrifugation to remove cellular debris. Alternatively, cellsof MSC346 could be lysed by a variety of methods that are well known(e.g. homogenization, detergent and/or enzymatic lysis, mechanicaldisruption, sonication and the like) and the chondroitin released bythese methods could be also be recovered in the supernatant followingcentrifugation. Chondroitin thus recovered could be further purified bymethods, such as alcohol precipitation, that are well known in the art.

Example 10

This example describes the construction of strains of E. coli K-12 thatcontain the chondroitin biosynthesis genes inserted in the chromosomeand demonstrates chondroitin production in these strains.

Examples 6-9 above describe chondroitin production in recombinant E.coli strains using plasmid vectors to introduce cloned genes that encodethe chondroitin biosynthesis proteins into heterologous host strains. Insome circumstances it could be desirable to introduce the clonedchondroitin biosynthesis genes into the chromosome of the recipient hoststrain. Placing the cloned genes in the chromosome eliminates therequirement for maintaining selective pressure to maintain theplasmid(s) which carry the chondroitin biosynthesis genes and could thuspotentially provide more stable expression strains or expression strainsthat are stable in the absence of selective pressure. Therefore, E. coliK-12 strains were constructed in which the E. coli K4 genes forchondroitin biosynthesis are stably integrated into the host chromosome.These “chromosomal expression strains” employ the Pm promoter and K4gene sets from pDD66 and pBR1052 integrated at the colanic acidbiosynthesis locus. The xylS regulatory gene also has been integratedinto the chromosome at a separate locus, the fhuA locus. Resultingconstructs were shown to produce high levels of chondroitin in shakeflasks and in fermentors (Examples 14 and 15).

Expression plasmids pDD66 and pBR1052 are described in Example 4. The K4chondroitin biosynthesis genes from pDD66 and pBR1052 were cloned intothe pMAK-CL replacement vector which is also described in Example 3.This vector, diagramed in FIG. 8L, contains cloned DNA regions upstreamand downstream of the colanic acid (CA) gene cluster and a unique AscIcloning site at the junction of these regions. As detailed in Example 3,this vector was used to construct a deletion of the entire CA genecluster in E. coli K-12 W3110 to generate strain MSC188. The K4 geneexpression cassettes were excised and gel-purified using the QIAEX IIGel Extraction Kit (QIAGEN Inc., Valencia, Calif.) according to thevendor protocol from pDD66 and pBR1052 as approximately 19 kb AscIfragments and these fragments were ligated with pMAK-CL DNA that wasAscI-digested, phosphatase-treated, and gel-purified. Transformants wereselected for resistance to tetracycline which is carried on the AscIfragments of pDD66 and pBR1052 along with the Pm promoters and theupstream and downstream transcription terminators. Derivatives ofpMAK-CL that contained the AscI fragments of pBR1052 or pDD66 wereidentified and designated pDD74 and pDD76, respectively. These plasmidsare diagramed in FIG. 8L.

Plasmids pDD74 and pDD76 were transformed into MSC188 to generatestrains MSC373 and MSC377, respectively, and derivatives of thesestrains in which the K4 gene set is integrated into the chromosome wereobtained as follows. MSC373 and MSC377 were grown overnight at 30° C. inLB+5 μg/mL tetracycline (Tc) and an aliquot of approximately 20 μL wasspotted and streaked onto an LB+Tc⁵ plate which was incubated overnightat 41° C. Colonies that arose on this plate were picked and re-streakedon LB+Tc⁵ plates at 43° C. Selection for maintenance of theplasmid-borne antibiotic resistance at high temperature selects forrecombination of the plasmid into the chromosome because the pDD74 andpDD76 plasmids, as derivatives of the pMAK705 vector, aretemperature-sensitive for replication (Hamilton et al., J Bact. 1989;171:4617-4622). Derivatives of MSC373 and MSC377 that were capable ofgrowth at 43° C. on tetracycline were sub-cultured overnight once inLB+Tc⁵ liquid medium at 30° C. and subsequently once overnight in LBliquid medium (without tetracycline) at 30° C. These overnight cultureswere then diluted and plated on LB plates at 30° C. and isolatedcolonies were toothpicked onto LB, LB+Tc⁵, and LB+34 μg/mLchloramphenicol (Cm³⁴) at 30° C. One tetracycline-resistant (TcR),chloramphenicol-sensitive (CmS) derivative was identified from each ofMSC377 and MSC373. These TcR, CmS derivatives, termed MSC391 and MSC392,respectively, were putative replacement strains in which recombinationhas occurred in such a way the K4 DNA sequence remains in the chromosomeat the CA locus while the remainder of the plasmid sequences have beenexcised by homologous recombination and the plasmid subsequently lost.PCR analyses of these isolates showed that the 5′ and 3′ ends of theapproximately 19 kb K4 DNA fragment are present in the expected locationwith respect to the chromosomal DNA sequences that flank the colanicacid locus.

As detailed in Example 4, DNA regions upstream and downstream of the E.coli fhuA gene were cloned by PCR, assembled and sequenced and thisdeletion fragment moved into the pMAK705 suicide plasmid to create areplacement vector for the fhuA locus termed pMAK705-ΔfhuA, or pDD73(FIG. 8M). The xylS regulatory gene was cloned into this replacementvector as follows. The xylS gene was excised from pDD42 as a PstIfragment and cloned into the PstI site of pDD73 to generate pDD77, whichis diagramed in FIG. 8N. The PstI fragment of pDD77 is identical to thexylS-containing PstI fragment present in expression plasmids pDD66 andpBR1052, and the parent vector pDD54. The K4 gene cluster replacementstrains, MSC391 and MSC392, described above were transformed with pDD77.Two isolates each from transformation of MSC391 and MSC392 by pDD77 wereselected and designated as follows:

MSC402 ≡MSC391 pDD77 “isolate A”MSC403≡MSC391 pDD77 “isolate B”MSC404 ≡MSC392 pDD77 “isolate A”MSC405 ≡MSC392 pDD77 “isolate B”

These strains were all tested in shake flasks for chondroitinbiosynthesis. Strains were grown in TB medium at 30° C.+Cm³ to selectfor maintenance the pDD77 plasmid. At OD A600 of approximately 0.2,cultures were induced by addition of 2 mM m-toluic acid (m-TA). Sampleswere taken at 24 and 48 hours post-induction and assayed forchondroitin. All four strains produced chondroitin. Results of theseassays are shown in Table 8A below. The chondroitin levels for inducedMSC404 and MSC405 were approximately 2.5-fold higher than MSC402 andMSC403. In this experiment, MSC404 and MSC405 produced about 65-70% theamount typically seen with MSC279 (MSC188 pDD66) in shake flasks underthese culture conditions; approximately 0.5 g/L. These results indicatethat recombinant E. coli containing a single chromosomal copy of the K4chondroitin biosynthesis genes is capable of producing significantamounts of chondroitin.

TABLE 8A Sample Strain description CH μg/mL OD A600 #1 MSC402 uninduced24 h MSC391 pDD77 11 11.6 isolate A #2 MSC402 + mTA 24 h MSC391 pDD77 4710.2 isolate A #3 MSC403 uninduced 24 h MSC391 pDD77 7 11.2 isolate B #4MSC403 + mTA 24 h MSC391 pDD77 47 10.0 isolate B #5 MSC404 uninduced 24h MSC392 pDD77 64 11.4 isolate A #6 MSC404 + mTA 24 h MSC392 pDD77 1149.4 isolate A #7 MSC405 uninduced 24 h MSC392 pDD77 16 11.7 isolate B #8MSC405 + mTA 24 h MSC392 pDD77 137 9.7 isolate B #9 MSC402 uninduced 48h MSC391 pDD77 29 15.9 isolate A #10 MSC402 + mTA 48 h MSC391 pDD77 15215.7 isolate A #11 MSC403 uninduced 48 h MSC391 pDD77 23 15.6 isolate B#12 MSC403 + mTA 48 h MSC391 pDD77 111 15.6 isolate B #13 MSC404uninduced 48 h MSC392 pDD77 142 15.7 isolate A #14 MSC404 + mTA 48 hMSC392 pDD77 344 14.9 isolate A #15 MSC405 uninduced 48 h MSC392 pDD7733 15.3 isolate B #16 MSC405 + mTA 48 h MSC392 pDD77 329 15.4 isolate B

The strains (MSC404 and MSC405) derived from pBR1052 appeared to be moreproductive than the strains derived from pDD66, but both chromosomalgenes sets functioned sufficiently well to produce chondroitin. Thechromosomal K4 gene set derived from pBR1052 contains a second copy ofthe Pm promoter inserted immediately upstream of the kpsF gene. Thisadded promoter was shown to enhance expression of the downstream genes(kpsFEDUCS) in the plasmid pBR1052 relative to expression in pDD66. Itis possible that the additional Pm promoter also increases downstreamgene expression in the chromosomal context and that the enhancedexpression of these genes can significantly increase CH production.

Derivatives of MSC403 and MSC405 were obtained in which theplasmid-borne xylS gene of pDD77 was integrated into the chromosome atthe fhuA locus using the two step “pop-in/pop-out” method as follows.MSC403 and MSC405 were grown overnight at 30° C. in LB+Cm³⁴. Thesecultures were diluted 10⁴-fold and 0.1 mL aliquots were plated onLB+Cm³⁴ at 43° C. After overnight incubation approximately 100 coloniesof varying size were obtained. Isolated colonies were picked andstreaked onto LB+Cm³ plates and grown overnight at 43° C.

Isolated colonies from these platings were picked and used to inoculate5 mL cultures of LB without any antibiotic. These cultures were grownovernight at 30° C. and subsequently twice passaged by 1000-folddilution and overnight growth in LB at 30° C. This third passage wasthen diluted 10⁶-fold and 0.1 mL aliquots were plated on LB at 30° C.and 37° C. Individual colonies from these platings were toothpicked ontoLB and LB+Cm³⁴ to test for loss of the plasmid.Chloramphenicol-sensitive (CmS) isolates were readily obtained, andthese were screened by PCR to identify the desired “pop-out” replacementstrains in which recombination occurred in such a way that thexylS-containing DNA sequence remained in the chromosome at the fhuAlocus while the remainder of the plasmid sequences were excised and theplasmid subsequently lost. This event also results in deletion of theentire fhuA gene from the E. coli chromosome. PCR analyses of theseisolates indicated that the 5′ and 3′ ends of the xylS DNA fragment noware present in the expected location with respect to the chromosomal DNAsequences that flank the fhuA locus. The resulting strains, MSC410,derived from MSC403, and MSC411, derived from MSC405, contain the xylSgene inserted at the fhuA locus and retain the K4 genes inserted at thecolanic acid locus.

MSC410 and MSC411 were tested for chondroitin biosynthesis. Strains weregrown in TB medium at 30° C. and at OD A600 of approximately 0.2,cultures were induced by addition of 2 mM m-TA. Samples were taken at 24and 48 hours post-induction and assayed for chondroitin. As shown belowin Table 8B, all of these strains produced very low levels ofchondroitin.

TABLE 8B Sample CH μg/mL OD A600 MSC410 uninduced 24 h 1.5 10.8 MSC410 +mTA 24 h 2.4 9.3 MSC411 uninduced 24 h 2.0 11.6 MSC411 + mTA 24 h 5.910.7 MSC410 uninduced 48 h 3.7 16.5 MSC410 + mTA 48 h 5.2 14.7 MSC411uninduced 48 h 4.4 15.4 MSC411 + mTA 48 h 12.2 16.1

This low productivity was unexpected, given that the immediatepredecessor strains produced significant quantities of chondroitin atcomparable cell densities under the same culture conditions. A priori,these results could indicate that the chromosomal insertion of the xylSgene does not produce a sufficient quantity of XylS protein to activatethe Pm promoter even in the presence of the inducer m-TA. Alternatively,the presumed DNA structures of the inserted K4 and/or xylS genes inthese strains might not be correct. The linkage relationships of the 5′and 3′ ends of both segments with respect to the chromosomal sequencesflanking the homologous colanic acid locus and fhuA locus sequences wereverified by PCR. However, those data alone do not confirm the precisestructures and sequences of the inserted DNAs in these strains.Rearrangement, deletion or mutation within the K4 or xylS DNA segmentscould have occurred in the course the derivation of MSC410 and MSC411from their respective CH-producing parents (MSC403 and MSC405) and couldhave resulted in impairment of CH biosynthesis.

Experiments were performed to test these hypotheses. Plasmid pDD77 wastransformed into MSC410 and MSC411 to test for the functionality of thechromosomal K4 gene sets in these two strains. The resulting strainswere designated MSC436 (MSC410 pDD77 “isolate A”), MSC437 (MSC410 pDD77“isolate B”). MSC438 (MSC411 pDD77 “isolate A”) and MSC439 (MSC411 pDD77“isolate B”). These strains were grown in TB medium at 30° C. and at ODA600 of approximately 0.2, cultures were induced by addition of 2 mMm-TA. Samples were taken at 24 and 48 hours post-induction and assayedfor chondroitin. As shown below in Table 8C, these four strains producedsignificant titers of chondroitin very similar to titers seen for theirantecedent strains MSC403 and MSC405 and much higher than theirimmediate predecessor strains MSC410 and MSC411. These results indicatedthat the defect in chondroitin biosynthesis in strains MSC410 and MSC411does not result from a defect in the K4 chondroitin biosynthesis genesper se.

TABLE 8C CH OD Strain description Sample μg/mL A600 MSC403 ≡ MSC391 +pDD77 #11 uninduced 48 h 23 15.6 (isolate B) #12 +mTA 48 h 111 15.6MSC410 ≡ MSC403 derivative #29 uninduced 48 h 4 16.5 having xylS in thechromosome #30 +mTA 48h 5 14.7 MSC436 ≡ MSC410 + pDD77 #53 uninduced 48h 16 16.3 (isolate A) #54 +mTA 48h 124 15.3 MSC437 ≡ MSC410 + pDD77 #55uninduced 48 h 14 16.0 (isolate B) #56 +mTA 48h 143 15.4 MSC405 ≡MSC392 + pDD77 #15 uninduced 48 h 33 15.3 (isolate B) #16 +mTA 48h 32915.4 MSC411 ≡ MSC405 derivative #31 uninduced 48 h 4 15.4 having xylS inthe chromosome #32 +mTA 48 h 12 16.1 MSC438 ≡ MSC411 + pDD77 #57uninduced 48 h 30 16.2 (isolate A) #58 +mTA 48 h 331 15.0 MSC439 ≡MSC411 + pDD77 #59 uninduced 48 h 26 16.4 (isolate B) #60 +mTA 48 h 20914.0

These findings suggest that chondroitin biosynthesis in strains MSC410and MSC411 could be low due to a defect in XylS protein functionresulting from some structural error in the gene coding sequence thatcould possibly have occurred during the generation of MSC410 and MSC411(from MSC403 and MSC405, respectively) or, alternatively, the xylS genesequence might be correct, but the expression level of the chromosomalxylS gene in these constructs might be insufficient to achieve optimalexpression of the K4 genes.

The xylS genes the in chromosomes of MSC410 and MSC411 were sequenced totest these hypotheses. The region of the E. coli chromosome containingthe xylS gene insertion was amplified by PCR using primers that flankthe integration site and the amplified DNA segment was sequenced. Thesequence of the xylS promoter and coding regions exactly matches theexpected sequence. This result suggested that the defect in xylSfunction in MSC410 and MSC411 is due to insufficient expression of theXylS protein from the chromosomal gene. Therefore, experiments wereperformed to enhance expression of the xylS gene. Toward that end, asynthetic optimized version of the xylS gene promoter, ribosome bindingsite, and 5′ untranslated region (UTR) was designed and synthesized, andthose modified sequences were introduced into the xylS replacementvector pDD77 and subsequently into the chromosome.

The synthesized fragment contains 134 bp of sequence matching theBipI-PsiI sequence of pDD77 followed by 86 bp of synthetic sequence upto the ATG start codon of xylS and extends 37 bp further into xylScoding sequence through the unique BglIII site. The sequence from theATG through the BglII site matches the sequence present in pDD77. TheBlpI-BglII fragment can be readily intrxiduced into pDD77 because theserestriction sites are unique in this plasmid. The 86 bp syntheticsequence (shown below) (SEQ ID NO:98) includes a consensus E. colipromoter (based on Hawley and McClure, Gene 1983; 11:2237-2255) and aconsensus Shine-Dalgarno (S-D) sequence (Shine and Dalgarno Proc. Natl.Acad. Sci. USA. 1974:71:1342-6). The sequence also incorporates astem-loop structure (indicated by underlined text) at the 5′ end of thepredicted mRNA. All of these features are expected to promote efficientexpression of the XylS protein.

The BlpI-BglI fragment (SEQ ID NO:140) was synthesized by a commercialvendor (DNA2.0) and the synthetic DNA containing the modified sequenceswas cloned into the xylS replacement vector, pDD77, as a BlpI-BglIIfragment in place of the native BlpI-BglII fragment containing thenative xylS regulatory sequences. The plasmid containing the modifiedxylS, termed pDD79 (FIG. 8N), was transformed into MSC392 to test theability of the modified xylS gene to activate the Pm promoter and drivechondroitin production. Three MSC392 transformants containing pDD79 werepicked and designated MSC458, MSC459 and MSC460. These strains, alongwith the MSC392 parent, were tested for chondroitin production in thestandard shake flask experiment. Strains were grown in TB medium(MSC392), or TB+Cm³⁴ (MSC458-460), at 30° C. and, at OD A600 ofapproximately 0.2, cultures were induced by addition of 2 mM m-TA.Samples were taken at 24, 48 and 72 hours post-induction. The 48 hoursamples were assayed for chondroitin. All of the pDD79-containingstrains produced approximately 300 μg/mL in both induced, and uninduced,cultures. In contrast, MSC392, lacking a xylS gene, produced only 4μg/mL chondroitin in both induced, and uninduced, cultures. Theseresults are shown below in table 8D.

TABLE 8D Strain description Sample CH μg/mL OD A600 MSC392 ≡ K4 gene setin #69; uninduced 48 h 4 15.9 chromosome #70; +mTA 48 h 4 16.0 MSC458 ≡MSC392 + #71; uninduced 48 h 314 14.8 pDD79 (modified xylS) #72; +mTA 48h 277 15.0 MSC459 ≡ MSC392 + #73; uninduced 48 h 325 13.9 pDD79(modified xylS) #74; +mTA 48 h 299 15.1 MSC460 ≡ MSC392 + #75; uninduced48 h 355 13.9 pDD79 (modified xylS) #76; +mTA 48 h 279 15.4

The observed value of approximately 300 μg/mL chondroitin is similar totiters seen for induced cultures of MSC405 and MSC438, which contain thenative xylS gene on plasmid pDD77. However, both uninduced and inducedcultures of MSC458, MSC459 and MSC460 produced essentially equivalent CHtiters. This result is consistent with increased production of XylS bythe modified xylS gene of pDD79 because overproduction of XylS has beenreported to activate the Pm promoter in the absence of any added inducer(Dominguez-Cuevas et al., J Bact. 2008; 190:3118-3128).

To test the functionality of the modified xylS gene when inserted intothe chromosome, derivatives of MSC459 were obtained in which theplasmid-borne xylS gene of pDD79 was integrated into the chromosome atthe jhuA locus using the two step “pop-in/pop-out” method as detailedabove in Example 3. MSC459 was grown overnight at 30° C. in LB+34 μg/mLchloramphenicol (Cm³⁴) and plated on LB+Cm³ at 43° C. After overnightincubation, isolated colonies were picked and streaked onto LB+Cm₃₄plates and again grown overnight at 43° C. Isolated colonies from theseplatings were picked and tested by colony PCR to confirm integration ofthe plasmid into the chromosome.

Two colonies that tested positive by PCR were used to inoculate 5 mLcultures of LB without any antibiotic. These cultures were grownovernight at 30° C. and subsequently passaged by 1000-fold dilution andovernight growth in LB at 30° C. These cultures were then diluted10⁶-fold and 0.1 mL aliquots were plated on LB at 37° C. Individualcolonies from these platings were toothpicked onto LB and LB+Cm³⁴ totest for loss of the plasmid. Chloramphenicol-sensitive (CmS) isolateswere readily obtained and 6 such isolates from each culture werescreened by PCR to identify the desired “pop-out” replacement strains inwhich recombination occurred in such a way that the xylS-containing DNAsequence remained in the chromosome at the fhuA locus while theremainder of the plasmid sequences were excised and the plasmidsubsequently lost. This event also results in deletion of the entirefhuA gene from the E. coli chromosome. PCR analyses of these isolatesindicated that the 5′ and 3′ ends of the xylS DNA fragment now arepresent in the expected location with respect to the chromosomal DNAsequences that flank the fhuA locus. Two such strains, MSC466 andMSC467, derived from MSC459, should now carry the xylS gene (withsynthetic promoter) inserted at the fhuA locus and the K4 genes insertedat the colanic acid locus.

MSC466 and MSC467 were tested for chondroitin biosynthesis in shakeflasks. Strains were grown in TB medium at 30° C. and, at OD A600 ofapproximately 0.2, cultures were induced by addition of 0, 1, 2 or 4 mMm-TA. Samples were taken at 24, 48 and 72 hours post-induction.Chondroitin assay data from the 48 hour samples are shown below in Table8E. Both strains produced substantial titers of chondroitin (>400 μg/mL)when induced with 1 or 2 mM m-TA. Cultures induced with 4 mM m-TAproduced somewhat lower chondroitin titers. Uninduced cultures producedlesser amounts of chondroitin, approximately 160-170 μg/mL. Theseresults are consistent with the hypothesis that the modified xylS genehaving the synthetic promoter, optimized ribosome binding site and 5′UTR hairpin structure is more efficiently expressed than the native xylSgene and therefore is more effective at stimulating transcription of theK4 chondroitin biosynthesis genes by the Pm promoters. The chromosomalstrains MSC467 and MSC466 do not contain any plasmids carrying the K4chondroitin biosynthesis genes, or the regulatory xylS gene, and areboth capable of production of substantial quantities of chondroitin.

TABLE 8E Strain m-TA (mM) chondroitin μg/mL OD, A600 MSC466 0 171 15.5 1496 15.6 2 456 15.6 4 419 15.6 MSC467 0 163 14.7 1 460 15.5 2 450 15.1 4325 13.7

MSC467 contains the tetracycline-resistance gene derived from pDD74 (seeFIG. 8L) inserted in the chromosome immediately downstream (3′) of thekpsS gene of the chromosomal K4 gene cluster. (See FIG. 8T) In order toallow use of tetracycline-resistance encoded by plasmid-borne genes as aselection for the introduction and maintenance of certain plasmids, thischromosomal tetracycline-resistance gene was deleted from the chromosomeof MSC467, as detailed below, using the “pop in/pop out” proceduredescribed in Example 3. The resulting tetracycline-sensitive derivativeof MSC467 was designated MSC561. Construction of MSC561 was accomplishedas follows.

Approximately 900 base pairs of chromosomal sequence immediatelyupstream of the tetR gene in MSC467 and pDD74 were amplified using pDD74DNA as template and primers BLR476 and BLR478:

BLR476 (SEQ ID NO: 101) 5>CGTCAAGCTTGTGAACGCCTATAGCAGCTTG>3 BLR478(SEQ ID NO: 102) 5>CAGTGGCGCGCCGAGCGATGATAAGCTGTC>3

The resulting PCR product was digested with HindIII and AscI and ligatedwith pMAK-CL (described in Example 3 and FIG. 8L) plasmid DNA that hadbeen digested with HindIII and AscI and treated with AntarcticPhosphatase (New England BioLabs, Ipswich, Mass.) according to thevendor protocol. Ligation products were transformed into E. coli NEB5α(New England BioLabs, Ipswich, Mass.), and plasmid DNAs from resultingtransformants were screened for the presence of the cloned PCR fragmentby diagnostic restriction endonuclease digestions. The recombinantplasmid from one such transformant was designated as pBR1087 and used ingene replacement experiments to delete the tetR gene from the MSC467chromosome. The structure of pBR1087 is diagrammed in FIG. 8U. Thisplasmid was transformed into MSC467 with selection forchloramphenicol-resistance at 30° C., the permissive temperature forreplication of the pMAK705-based replicon. Cultures grown at 30° C. weresubsequently plated at 43° C. in the presence of chloramphenicol at 34μg/mL (Cm³⁴), and resulting colonies were picked and streaked ontoLB+Cm³⁴ plates at 43° C. Resulting colonies were screened by PCR for theintegration of pBR1087 at the targeted locus, and isolates that wereidentified as containing the plasmid sequences integrated at this locuswere sub-cultured in LB liquid medium at 30° C. in the absence ofchloramphenicol. Subsequently, isolates thus sub-cultured were plated onLB at 30° C. in the absence of chloramphenicol, and resulting colonieswere tested for sensitivity to chloramphenicol and tetracycline.Chloramphenicol-sensitive, tetracycline-sensitive derivatives, thepresumptive gene replacement strains in which the tetR gene has beendeleted as a result of excision of the integrated plasmid, were obtainedand screened by PCR to confirm this presumptive chromosomal structure.

One strain that was thus identified as having undergone deletion of thetetR gene was designated as MSC561 and the structure of the chromosomalK4 gene cluster of MSC561 is shown in FIG. 8T. The deletion extends from71 bp downstream of the kpsS coding sequence up to the AscI site at the5′ end of the sequences downstream of the colanic acid locus. Thedeletion includes the entire tetR gene.

Example 11

This example demonstrates that increasing the gene dosage of the K4region 2 genes (kfoABCFG) relative to the regions 1 and 3 genes resultsin significantly greater chondroitin production in E. coli K-12 shakeflask cultures.

E. coli K-12 strain MSC467 (Example 10) contains regions 1, 2, and 3 atthe colanic acid locus under control of the Pm promoter and xylS at thefhuA locus under control of the synthetic consensus promoter. PlasmidpCX039 (Example 4) contains the region 2 kfoABCFG genes under control ofthe Pm promoter and also contains the native xylS gene. pCX039 wastransformed into MSC467 to create strain MSC499. Control strain MSC498was created by transforming pDD63 (Example 4) into MSC467. Chondroitinproduction in shake flask cultures (TB/Cm³ medium, 30° C., 72 hr) withvariable inducer concentrations was determined for these two strains.(Table 9A).

TABLE 9A Strain Strain description m-TA CH; μg/mL MSC498 MSC467 + pDD63(xylS; 0 109 no K4 genes) 1 mM 349 2 mM 342 3 mM 359 MSC499 MSC467 +pCX039 (xylS; 0 760 kfoABCFG) 1 mM 1067 2 mM 916 3 mM 951

Increasing the region 2 gene dosages by presenting them on a plasmidclearly resulted in higher chondroitin production. A relatively highlevel of production was seen in strain MSC499 without induction. Thiswas likely due to uninduced expression of the K4 genes due to enhancedexpression of the modified xylS gene present in the MSC499 chromosome.As noted above, it is known that high levels of XylS protein canactivate the Pm promoter even in the absence of added inducer(Dominguez-Cuevas et al., 2008). To determine whether additionalplasmid-encoxled XylS is required for optimal chondroitin production inthis plasmid system, derivatives of plasmids pDD63 and pCX039 lackingxylS genes were constructed. These plasmids each contain two Nsi Irestriction sites that flank the entire xylS gene coding sequence within1049 bp fragments (see Example 4). Samples of these plasmids weredigested with NsiI, heat-treated to inactivate the enzyme, and thentreated with T4 DNA ligase to generate circular plasmids lacking thexylS gene fragment. The pDD63ΔNsiI was first transformed into E. coliMSC188 (Example 3). The characterized pDD63ΔxylS plasmid was namedpCX069. This plasmid was subsequently transformed into E. coli MSC467(Example 10) to create strain MSC510. The pCX039ΔNsi sample wastransformed directly into MSC467, and the characterized dxylS plasmidwas named pCX074. Chondroitin production by these strains plus theMSC498 and MSC499 control strains was determined as described previouslyin this Example, and the results are shown in Table 9B.

TABLE 9B strain plasmid m-TA CH; μg/mL MSC498 MSC467 + pDD63 (xylS; 1 mM338 no K4 genes) 2 mM 358 MSC499 MSC467 + pCX039 (xylS; 1 mM 1024kfoABCFG) 2 mM 964 MSC510 MSC467 + pCX069 1 mM 310 (pDD63ΔxylS) 2 mM 359MSC511 MSC467 + pCX074 1 mM 874 (pCX039ΔxylS) 2 mM 1135

Deletion of xylS from pCX039 did not reduce the maximal amount ofchondroitin produced but a higher inducer level was needed to achievethe maximal level; see MSC511 vs. MSC499 in Table 9B. This resultillustrates the interrelatedness of XylS levels, inducer levels, K4 genecomplement, and chondroitin productivity.

Example 4 above describes the derivation of plasmid pDD80, apMAK705-based replacement plasmid designed for insertion of xylS and theregion 2 kfoABCFG genes, all expressed from the synthetic consensuspromoter, into the E. coli chromosome at the fhuA locus. Table 9C belowdescribes chondroitin production in MSC467 (chromosomal xylS) and MSC392(no xylS) strains (Example 10) containing pDD80 as an extra-chromosomalelement.

TABLE 9C Strain Strain description m-TA CH; μg/mL MSC499 MSC467 + pCX039(xylS; 0 410 kfoABCFG) 1 mM 897 MSC522 MSC467 + pDD080 (xylS/ 0 997kfoABCFG) 1 mM 896 MSC526 MSC392 + pDD080 (xylS/ 0 868 kfoABCFG) 1 mM968

Similar to the cases with pCX039, plasmid pDD80 enhanced chondroitinproduction in E. coli host strains containing chromosomal copies of theentire complement of K4 chondroitin biosynthesis genes (regions 1, 2,and 3). Induction had little effect on chondroitin production in strainscontaining pDD80, regardless of the presence or absence of xylS copiesin the chromosome. This is likely a result of a relatively high level ofXylS in these strains due to expression, from a multicopy plasmid, ofthe modified xylS gene driven by the strong synthetic promoter, andcontaining the optimized ribosome binding site and hairpin structureadded to the mRNA 5-prime end.

Example 12

This example demonstrates that the addition of a single additional copyof the kfoABCFG genes to the chromosome of strain MSC467 increaseschondroitin production.

Example 11 above demonstrates that chondroitin production by strainMSC467 in shake flasks was greatly enhanced when extra copies of the K4region 2 genes kfoABCFG under control of the Pm promoter were introducedinto MSC467 on plasmid pCX039. Similar results were obtained whenplasmid pDD80 was introduced into MSC467. Plasmid pDD80, described inExample 4 carries the kfoABCFG genes under transcriptional control ofthe synthetic xylS promoter described in Example 10 above. These resultsindicate that increasing the levels of one or more of the proteinsencoded by the kfoABCFG genes significantly increases chondroitinproduction. Cloning of these genes on a multi-copy plasmid provides onemethod for increasing the production of these proteins. An alternativemethod for increasing the production of these proteins, withoutemploying a plasmid expression platform, is to insert multiple copies ofthese genes into the chromosome of the host organism.

A second copy of the kfoABCFG gene set was inserted into the chromosomeof the MSC467 at the fhuA locus, immediately downstream of the modifiedxylS gene, under transcriptional control of the synthetic xylS promoter.A replacement vector was constructed for this purpose by cloning thekfoABCFG genes on a PstI fragment excised from pCX039 into thecompatible NsiI site of pDD79 as detailed in Example 4. In the resultingplasmid, pDD80, the kfoABCFG genes are transcribed by the synthetic xylSpromoter which was designed to be a strong constitutive promoter. pDD80was transformed into MSC467 to produce strain MSC522 which was shown inExample 10 above to prodxuce approximately 1 g/L chondroitin in shakeflasks. A replacement strain (MSC537) was derived from MSC522 viaprocedures for pMAK705-based plasmid replacements as detailed in Example3 and Example 10 above. This strain carries one additional copy of thekfoABCFG genes inserted at the fhuA locus, immediately downstream of thexvIS gene, in the MSC467 chromosome.

MSC537 was tested in parallel with MSC467 for chondroitin production.Cultures were inoculated at approximately 0.01 OD A600 in TB medium at30° C. At an OD of approximately 0.10, cultures were induced by additionof 1 mM m-TA and cultured for an additional 72 hours. Samples were takenfor chondroitin assays at 48 and 72 hours post-induction. At 72 hourspost-induction, MSC537 produced 0.57 g/L chondroitin while MSC467 gave0.45 g/L; an increase of approximately 25% for MSC537 vs. MSC467. Infurther experiments, when MSC537 and MSC467 were tested in parallel forchondroitin production in shake flasks, MSC537 consistently producedmore chondroitin (20-30%) than MSC467. This result indicates that theaddition of a single additional copy of the K4 region 2 genes kfoABCFGto the MSC467 chromosome can increase chondroitin production althoughnot to the same extent that the addition of multiple copies of thekfoABCFG genes increases chondroitin production in strains MSC499 andMSC522. These two strains, which carry the cloned kfoABCFG genes onmulti-copy plasmids, produce approximately 2-fold more chondroitin thanMSC467 (see Example 10). The plasmid pCX039 was transformed into MSC537to create strain MSC551.

Like MSC467, the MSC537 strain contains the tetracycline-resistance genederived from pDD74 (see FIG. 8L) inserted in the chromosome immediatelydownstream (3′) of the kpsS gene of the chromosomal K4 gene clusterpresent in MSC537. As noted above, in some emtbodiments it is desirablefor strains to be tetracycline-sensitive. Therefore, the iteR gene wasdeleted from the chromosome of MSC537 using the same procedures, andreplacement plasmid (pBR1087) as described above in Example 10 fordeletion of the tetR gene from the chromosome of MSC467. The resultingtetracycline-sensitive derivative of MSC537 was designated MSC562. In ashake flask experiment, MSC562 and MSC537 were grown in TB medium at 30°C. and induced by addition of 1 mM m-TA. Chondroitin titers of culturesamples harvested at 72 hours post-induction were determined and foundto be comparable to each other; 0.51 g/L for MSC562 vs. 0.57 g/L forMSC537.

Plasmids pCX039, which contains the kfoABCFG genes as shown in FIG. 8Q,and pDD63, the vector-only control plasmid shown in FIG. 8I, weretransformed into strain MSC562 to create strains MSC564 and MSC563,respectively. In shake flasks experiments, MSC563 and MSC564 were grownat 30° C. in TB medium containing tetracycline (5 μg/mL) for plasmidselection and induced with 1 mM m-TA. At 72 hours post-induction,cultures of MSC564 and MSC563 produced chondroitin at titers of 0.81 g/Land 0.29 g/L, respectively.

In this experiment, in which cultures were grown in the presence oftetracycline, plasmid retention of pCX039 in MSC564 was very efficient.A sample from the 72 hour culture of MSC564 was diluted and plated on LBplates and LB plates containing 5 μg/mL tetracycline. The titers ofcolony forming units (CFU) were not significantly different under thesetwo plating conditions; 1.16×10⁹ CFU/mL on LB vs. 1.28×10⁹ CFU/mL onLB+tetracycline. Thus, under the conditions of this experiment, no lossof the plasmid was detected.

It is expected that subsequent additions of more copies of the kJoABCFGgenes to the chromosome of MSC537 will further increase chondroitinproduction in this strain. Additional copies of these genes can beinserted at other chromosomal loci using the gene targeting proceduresdetailed in Example 3. A wide variety of non-essential loci are known inE. coli which could serve as additional sites for integration of thesegenes. In addition, tandem arrays, consisting of two or more copies ofthe kjoABCFG gene set, could be constructed on gene replacement plasmidsand introduced into the chromosome in a single gene replacement event.

Moreover, additional methods for increasing the production of theproteins encoded by the kfoABCFG genes include codon optimization ofprotein coding sequences, and optimization of the sequences ofpromoters, ribosome binding sites and 5-prime untranslated regions ofmRNAs of these genes. Such sequence optimizations could be applied togenes that were expressed from plasmid vectors and to genes that wereinserted into the chromosome.

Example 13

This example describes the introduction of chondroitin biosynthesisgenes into Xanthomonas campestris using plasmid vectors and chromosomalIntegration, and demonstrates recombinant DNA mediated chondroitinbiosynthesis in Xanthomonas campestris in shake flasks.

Specifically, described herein is the construction of plasmidscontaining combinations of the K4 biosynthetic genes and theirintroduction into Xanthomonas campestris strain MSC255. Furtherdescribed is the use of derivatives of plasmids pKM001 and pKM002(described in Example 3) to stably insert the chondroitin biosyntheticgenes and subsets thereof into the X. campestris strain MSC255chromosome at the site of the deletion of the xanthan gum operon.

Introduction of Chondroitin Biosynthetic Genes into X. Campestris asExtra-Chromosomal Elements.

The present inventors have discovered that introduction of largeplasmids into X. campestris directly from E. coli donors (e.g., viatri-parental crosses) or as plasmid purified from E. coli strains (e.g.,via electroporation—see below) can result in structural anomalies in theresulting plasmid in X. campestris. Relatively small plasmids appearless sensitive to this phenomenon, which may be due to restrictionsystems native to X. campestris (Feyter and Gabriel, J. Bact.1991:173:6421-6427, da Silva et al., Nature 2002; 417:459, Roberts etal., Nuc. Acid Res. 2010; 38:D234) that have a relatively greater effecton larger DNA molecules. The present inventors have used two approachesthat successfully overcome this effect. In one approach, a large plasmidencompassing regions 1, 2, and 3 genes was reconstructed from smallerplasmids purified from X. campestris transformants. In a secondapproach, the regions 1, 2, and 3 genes were split between two (smaller)compatible plasmids.

Electroporation was used to introduce plasmids into X. campestris cells(Oshiro et al, J. Microbiol. Method 65:171-179, 2006). Plasmid pDD67(described in Example 4) was digested with restriction enzymes BamHI andRsrII (which bound the region 2 genes) followed by reaction with T4 DNApolymerase (to create blunt ends) and ligation. The resulting mix wastransformed into E. coli, and tetracycline-resistant isolates werecharacterized. The resulting plasmid, pKM005, shown in FIG. 10A,containing Pm-driven regions 1 and 3 genes, was then transformed byelectroporation into X. campestris to create strain MSC338. Likewise,pCX039 (Example 4) was transformed into X. campestris to create MSC326.Plasmids pKM005, purified from MSC338, and pCX039, purified from MSC326,were each digested with HindIII plus AvrII, and the region 1, 3 fragmentfrom pKM005 was ligated with the vector/region 2 fragment of pCX039. Theresulting mix was transformed directly into MSC255 with selection fortetracycline-resistance. One X. campestris transformant, MSC348, wasshown to contain the plasmid pKM007 (FIG. 10A) which wasindistinguishable from pDD67 by restriction digests of plasmid that hadbeen transferred back to E. coli. For control purposes, X. campestrisstrain MSC255 was transformed with pDD63 vector to create strain MSC397.

Plasmid pJAK15 (ATCC77290, obtained from ATCC) belongs to the IncQincompatibility group and encodes kanamycin resistance. TheHindIII/AvrII fragment from pKM005 (containing the Pm-driven regions 1and 3; see above) was ligated with the vector/kanamycin resistanceHindIII/XbaI fragment from pJAK15. The resulting plasmid, pKM006, asshown in FIG. 10B, contains inducible regions 1 and 3 genes on a vectorcompatible with the various pBHR1-derived plasmids. Strain MSC326(MSC255 pCX039) was transformed with pKMOO6 with simultaneous selectionfor kanamycin and tetracycline resistance to create strain MSC350.

The knock-out vectors pKM001 and pKM002 were first modified byintroduction of a short DNA oligonucleotide linker that carries AscI,SbfI, SwaI and XhoI cloning sites into the NotI restriction sites thatdefine the junctions of the upstream and downstream regions of homologyin pKM001 and pKM002. The linker was made from annealed oligonucleotidesprKM015 and prKM016 such that single-strand overhangs were present thatare compatible with those generated by NotI digestion of pKM001 andpKM002. The resulting plasmids, designated pKM008 and pKM009respectively, contain the linker oriented such that the AscI restrictionsites are proximal to the upstream regions.

prKM015 (5′-3′) (SEQ ID NO: 99)         AscI           SwaI  XhoIGGCCGCGGCGCGCCTGCAGG 

CTCGAGGC prKM016 (3′-5′) (SEQ ID NO: 100)CGCCGCGCGGACGTCCTAAATTTAGAGCTCCGCCGG SbfI

Replacement vectors having only the K4 region 2 genes (kfoA, kfoB, kfoC,kfoF and kfoG) were constructed as follows: pKM008 was digested withSbfI-XhoI or AscI-XhoI and pCX039 was digested with SbfI-SalI orAscI-SalI. SbfI-SalI treatment produces a kfoABCFG-containing fragmentfrom pCX039 and AscI-SalI treatment produces a Pmpromoter-kfoABCFG-containing fragment. These fragments were cloned intopKM008 which was first digested with SbfI-XhoI or AscI-XhoI to generateplasmids pKM010 (kfoABCFG: Pm−) and pKM011 (kfoABCFG: Pm+) respectively.Plasmids pKM010 (SEQ ID NO:145) and pKM011 (SEQ ID NO:146) were used toconstruct additional replacement vectors that also incorporate the K4region 3 (kpsF, kpsE, kpsD, kpsU, kpsC and kpsC) and region 1 (kpsM andkpsT) genes. pKM010 and pKM011 were digested with SbJl-AvrII and theselinearized vectors were ligated with the kpsFEDUCSMT-containing fragmentproduced by SbJl-AvrII digestion of pDD67. The resulting plasmids weredesignated pKM012 (Pm−) and pKM013 (Pm+). Derivation of plasmids pKM010(SEQ ID NO:145) and pKM012 (SEQ ID NO:147) is diagrammed in FIG. 10C.Derivation of plasmids pKM01 (SEQ ID NO:146) and pKM013 (SEQ ID NO:148)is diagrammed in FIG. 10D.

The upstream region of the xanthan gum gene cluster, which is cloned inall of the pKM008-pKM013 constructs, is reported to encompass the gumgene cluster promoter (Federico et al; J. Bact. 1996:178:4313-4318).Therefore, X. campestris strains that contain sequences derived frompKM010 or pKM012 recombined into the chromosome are expected totranscribe the K4 genes from the endogenous gum promoter. In contrast,pKM011 and pKM013 have terminator sequences (derived from pDD67) locatedbetween the gum promoter and the Pm promoter. Therefore, the expressionof K4 genes in recombinant X. campestris containing pKM011 or pKM013sequences is expected to be regulated by the Pm/XylS system.

For each of these replacement plasmids, the “pop-in/pop-out” methodology(detailed above) was used to recombine the respective K4 gene clustersinto the chromosome of X. campestris strain MSC255 at the (deleted) gumlocus. The plasmids were introduced into MSC255 by electroporation withselection for tetracycline resistance. Intermediate and final strainsare indicated in Table 10A below. Recombinants in which the “pop-in” hastaken place at the gum locus were identified by PCR. Resolution of theintegrants was allowed to occur by culturing in the absence ofantibiotic selection, and tetracycline sensitive (“pop-out”) derivativeswere subsequently identified. PCR was then used to identify the“pop-out” derivatives in which the respective K4 gene clusters havesuccessfully integrated into the gum locus in the desired orientation.Plasmid pDD63 (carrying the xylS inducer gene) was then transformed intothe recombinants in which the Pm promoter is driving the K4 gene sets.The genetic structure of the four key Xanthomonas chromosomal insertionstrains (containing no plasmid-encoded K4 genes). MSC480, MSC469,MSC461, and MAC494, is summarized in Table 10A.

TABLE 10A Replacement pDD63 (xylS) plasmid Promoter K4 genes “pop-in”strain “pop-out” strain transformant pKM010 gum kfoABCFG MSC435 MSC480 —pKM011 Pm kfoABCFG MSC441 MSC455 MSC469 pKM012 gum kpsFEDUCS MSC466MSC461 — kpsMT kfoABCFG pKM013 Pm kpsFEDUCS MSC448 MSC493 MSC494 kpsMTkfoABCFGChondroitin Production in Shake Flasks of Recombinant Xanthomonascampestris.

Unless otherwise stated, growth of X. campestris strains in shake flasksfor evaluation of chondroitin production was conducted in YMG medium (5g/L proteose peptone, 3 g/L yeast extract, 3 g/L malt extract, 10 g/Lglucose). Cultures were routinely grown at 30° C. in 50 mL medium in a250 mL growth flask at 200-225 rpm with the required antibiotics forselection (e.g., 2-5 μg/mL tetracycline, 10 μg/mL kanamycin) for 48 hr.For induction of Pm-driven gene sets, 2 mM m-TA was added when culturedensities reach approx. OD600=0.5.

Strains with Extra-Chromosomal K4 Biosynthetic Genes.

Chondroitin production in Xanthomonas campestris strain MSC255(ΔgumB−gumM) transformed with various plasmids was determined byculturing and assaying as described above. Results are shown in Table10B.

TABLE 10B K4 Culture CH Strain Plasmids genes age (hr) OD600 (μg/mL)MSC397 pDD63 — 48 5.44 nd* MSC326 pCX039 region 2 48 1.53 104 MSC348pKM007 all 48 2.49 37.2 MSC350 pCX039 all 41 2.16 41.2 pKM006 *nonedetected

The control strain MSC397, containing the empty vector, made nodetectable chondroitin. Strains MSC348 and MSC350, containing theregions 1, 2, and 3 genes, produced about 40 μg/mL chondroitin underthese conditions. Strain MSC326, containing just the region 2 genes,produced about 100 μg/mL chondroitin.

In another experiment, chondroitin production in strain MSC326 (MSC255pCX039) containing the kfoABCFG genes was 166 μg/mL after 48 hr; controlstrain MSC397 (MSC255 pDD63—vector control) produced no detectablechondroitin. In fractions from non-autoclaved samples of the MSC326culture, the cell-free supernate and cell pellet fractions contained 100μg/mL and 71 μg/mL chondroitin, respectively. These results show thatthe K4 region 2 genes (minus those directing fructosylation) aresufficient for chondroitin production in X. campestris and furthersuggest that the chondroitin is being exported from the cells by someuncharacterized endogenous mechanism or by cell breakdown or lysis.

Strains with Chromosomally Encoded K4 Biosynthetic Genes.

The extra-chromosomal plasmids described above are, with varyingfrequencies, lost from bacterial cells in culture as defined by loss ofantibiotic resistance. Integration of the K4 biosynthetic genes into theX. campestris chromosome should minimize such instability as well asfacilitate large scale culturing of these strains. Chondroitinproduction in the four strains with chromosomally integrated K4 genes(see above) is shown in the Table 10C below. The strains were grown ineither modified YMG medium (YMGM(5): YMG buffered with 80 mM MOPS (pH7.0), 5 g/L glucose) or TB medium for 48 hours. Tetracycline is presentat 5 μg/mL in strains with pDD63. Chondroitin was produced from bothpromoters and in both media. Significantly, chondroitin was producedwhen only the region 2 genes are present. See table 10C.

TABLE 10C chon- mTA droitin Strain Promoter K4 genes Medium inducer(μg/mL) MSC480 Gum region 2 YMGM (5) none 97.5 MSC480 Gum region 2 TBnone 117 MSC469 Pm (+pDD63) region 2 YMGM (5) 2 mM 316 MSC469 Pm(+pDD63) region 2 TB 2 mM 297 MSC461 Gum all YMGM (5) none 80.8 MSC461Gum all TB none na* MSC494 Pm (+pDD63) all YMGM (5) 2 mM 108 MSC494 Pm(+pDD63) all TB 2 mM 122 *not assayed

In another experiment, selected strains were grown in YMGM medium withvarying glucose levels. Chondroitin was produced at all glucoseconcentrations in strains carrying sets of the biosynthetic genes (butnot in a control strain lacking such genes). The greatest productionunder these conditions was 390 μg/mL in strain MSC469 containing justthe region 2 genes under control of Pm/xylS induction. Assay of thenon-autoclaved cell-free supernate and cell pellet of the YMGM+10 g/Lglucose culture of MSC469 found 167 μg/mL and 150 μg/mL chondroitin,respectively. As before, these results suggest that the chondroitin isbeing exported from the cells. See Table 10D.

TABLE 10D Glucose chondroitin Strain Promoter K4 genes Medium (g/L) mTAinducer OD600 (μg/mL) MSC255 — — YMGM 10 none 4.660 nd* MSC461 Gum allYMGM 5 none 2.492 69.2 MSC461 Gum all YMGM 10 none 2.353 62.5 MSC461 Gumall YMGM 20 none 2.278 72.9 MSC469 Pm region YMGM 5 2 mM 3.515 390.2(pDD63) 2 MSC469 Pm region YMGM 10 2 mM 3.586 367.4 (pDD63) 2 MSC469 Pmregion YMGM 20 2 mM 2.893 246.7 (pDD63) 2 *none detected

Example 14

This example describes the methods for assaying fructosylated andunfructosylated chondroitin.

Preparation of Chondroitin from Bacteria

Recombinant chondroitin (rCH) was captured using an anion-exchangerDEAE-cellulose DE52 column. After washing with 5 volumes of 100 mM NaCl,the column was eluted with 5 volumes of 300 mM NaCl. The eluate wasconcentrated and dialyzed against 10 volumes of distilled water. Thedialyzed solution was lyophilized. The lyophilized powder was used asrCH.

Fructosylated chondroitin capsular polysaccharide (K4P) was purifiedfrom the culture of strain U1-41 (Escherichia coli 05:K4:H4) accordingto the method of Manzoni, M. et al. (Biotechnology Letters1996:18:383-386). Preparation of defructosylated K4P (DFK4P) from K4Pwas performed according to the method of Lidholt, K., et al. (J. Biol.Chem. 1997; 272: 2682).

Sample Preparation and Chondroitin Assay by HPLC

Flask culture samples (typically 5 mL) were autoclaved at 121° C. at >15psi for 5 min. and allowed to cool. Samples were then re-adjusted to theoriginal volume with water as needed to account for losses duringautoclaving. Samples (1.5-5 mL) were centrifuged (typically 3500 g for10 min. for lower cell density flask cultures; 12000 g for 5 min. forfermentation or higher density cultures) to yield supernate and pelletfractions. In some cases, as indicated, samples were centrifuged withoutprior autoclaving. Culture samples or separated supernates and pelletswere usually stored at −20° C. until assayed.

For analysis of cell-associated chondroitin, cell pellets wereresuspended in an original volume of 50 mM sodium phosphate buffer pH7.2 and hydrolyzed with 5-10 mg/mL lysozyme (Sigma L-7651) and 60 U/mLdeoxyribonuclease I (Sigma D-4527) at 37° C. for 2 hr followed by 200μg/mL proteinase K (Promega V3021) at 37° C. for 1 hr. After terminatingthe reaction (90° C., 5 min), the solutions were centrifuged to removecell debris.

For analysis of fructosylated chondroitin capsular polysaccharide (K4P),samples (flask/fermentation broth supernates or clarified hydrolyzedcell pellets) were first defructosylated using mild acid hydrolysis;i.e., adjusted with HCl to pH 1.5, incubated at 80° C. for 30 min. andthen neutralized with 0.5 M sodium carbonate. Prior to lyophilization,the DFK4P samples and the non-fructosylated rCH samples (fermentationbroth supernates, hydrolyzed cell pellets or reconstituted precipitates)were dialyzed overnight against deionized water (Pierce BiotechnologySlide-A-Lyzer®, molecular weight cut-off 7 kD) or partially purified bycentrifugal ultra-filtration (Amicon Ultra-0.5 Centrifugal FilterDevice, 10 kD nominal molecular weight cut-off) with elution indeionized water.

Chondroitin that is not fructosylated can be completely hydrolyzed to anunsaturated non-sulfated disaccharide,2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronicacid)-D-galactose (Δdi-0S) by the chondroitin-degrading enzyme, whichwas named chondroitinase ABC (Seikagaku Biobusiness, Japan).Consequently, the amount of chondroitin in sample solutions that is notfructosylated can be determined by quantifying this disaccharideenzymatically produced from the polysaccharide using a HPLC system.

The residue after lyophilization was dissolved in THB (50 mM Tris-HClbuffer with 50 mM sodium acetate pH 8.0) and hydrolyzed withchondroitinase ABC (2 units/mL, 37° C. for 3 hr). After terminating theenzymatic reaction by heating at 90° C. for 5 min., the mixture wascentrifuged at 10000 rpm for 5 min. to remove insoluble sediments. Thesupernatant was filtrated using Microcon centrifugal filter (UltracelYM-10; Millipore) to remove enzyme and non-chondroitin polysaccharides.The resulting unsaturated disaccharide(2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronicacid)-D-galactose; Δdi-0S) was separated by reverse-phase ion-pair HPLC(Senshu Pak Docosil, 4.6×150 mm; particle size, 5 im), labeledpost-column with 2-cyanoacetamide, detected with fluorescence (Toyoda,H., et al. J. Biol. Chem., 2000; 275:2269), and quantified againstexternal standards prepared from commercially available chondroitin(Seikagaku Biobusiness, Japan) or rCH. The typical calibration curve forthe disaccharide is shown in FIG. 11B. The calibration curve for thedisaccharide was linear in the range of 2 to 200 μg/mL, and thedetection limit of the disaccharide was 1 μg/mL. The concentration ofthe chondroitin polysaccharide can be calculated using the followingformula,

Concentration; μg/mL=[A]/[S]×[D],

where [A] represents the peak area of Δdi-0S in the sample chromatogram,[S] represents the slope of calibration curve for Δdi-0S concentrationand [D] represents the dilution factor.

ELISA Method for Quantification of Fructosylated Chondroitin

Biotinylation coupling through carboxyl groups of K4P was performedaccording to the method of Osmond, R. I. W. et al., AnalyticalBiochemistry 2002; 310: 199-207). 100 μl of biotinylated K4P (1 μg/ml)was conjugated to a streptoavidin coated 96-well micro-titer-plate(Thermo Scientific, Japan) at room temperature for 30 min. After washingthe plate using 50 mM Tris-HCl buffered saline (NaCl 100 mM)supplemented with 0.05% TWEEN® 20 and 0.05% Proclin (pH 7.5). 50 μl ofculture supernatant or standard K4P solution, and 50 μl of anti-K4Pserum (Statens Serum Institut, Denmark) at a dilution of 2.5×10⁶ wereadded to the wells and incubated for 60 min. After washing the wellsagain, HRP-labeled anti-rabbit immunoglobulins (P0448, DAKO JAPAN,Japan) at a dilution of 2000× was added and incubated for 60 min. Afterwashing the wells again, TMB solution (TMBW-1000-01, BioFX LaboratoriesInc., Owings Mills, Md.) containing H₂O₂ was added as substrate andincubated at room temperature for 30 min. 50 ml of Stop Reagent(STRP-1000-01, BioFX Laboratories Inc., Owings Mills, Md.) was added andthe absorbance at 450 nm was measured. Under these assay conditions,other polysaccharides such as unfructosylated chondroitin, heparosan andDFK4P do not compete with K4P. A typical standard curve is shown in FIG.11A.

SEC-HPLC Analysis of rCH, K4P and DFK4P

Weight average molecular weight (“Mw”) and chondroitinase-digestibilityof polysaccharides was determined by analyzing on a SEC-HPLC using anTOSOH HLC-8220GPC system equipped with a refractive index detector andtandem columns of TSK-gel PWXL-4000, PWXL-3000, and PWXL-2500 (TOSOH,Japan) at a constant flow rate of 0.6 mL/min with 0.2 M NaCl. Fifty μLof polysaccharide solution was injected at a concentration of 1 mg/mL onthe column. The columns and detector compartment were maintained at 40°C. Mw-defined chondroitin sulfates (Mw: 52.2, 31.4, 20.0, 10.0, 6.6, and1.0 kDa) were used as molecular weight standards.

Typical elution profiles of rCH (which is not fructosylated) before andafter chondroitinase ABC digestion are shown in FIG. 12. Calculatedweight average molecular weight of rCH was 120 kDa.

Determination of chondroitinase digestibility of K4P and DFK4P wasperformed as follows. K4P and DFK4P were dissolved in 50 mM THB(Tris-HCl with 50 mM sodium acetate pH 8.0), to give a finalconcentration of 1 mg/mL and divided into equal parts. A part of thesolution was directly analyzed on the SEC-HPLC as described above.Another part was analyzed on the same system followed by processing withchondroitinase ABC (final concentration; 2 units/mL) at 37° C. for 3hours. Results are shown in FIG. 13. The molecular weight values of K4Pand DFK4P prepared from the culture of E. coli K4 U1-41 were 33 kDa and28 kDa, respectively. DFK4P was completely digested into thedisaccharide, Δdi-0S, by chondroitinase treatment whereas K4P waspartially digested with the enzyme (FIG. 13). These results indicatedthat K4P was converted to the defructosylated form without affecting thechondroitin backbone structure of K4P by the defructosylation processdescribed above. Consequently, the amount of K4P in samples can also bedetermined by quantifying the disaccharide enzymatically produced fromthe polysaccharide using the chondroitinase/HPLC method when sampleswere processed in the defructosylation process before the enzymedigestion.

Example 15

This example illustrates sulfation of chondroitin.

Chondroitin prepared in Example 14 was subjected to partialdepolymerization to obtain a chondroitin of molecular weight app. 30kDa. 30 mg of this chondroitin was solubilized into 0.6 mL of dryformamide (FA) with stirring at 60° C. When the solution becamecompletely homogeneous, solid sulfur trioxide-TEA complex (5 eq. ofchondroitin disaccharide unit) was added and the stirring was continuedfor 120 min. The sulfation reaction was stopped by addition of 3× vol.of 1 M sodium acetate solution and was allowed to stand for another 30min at room temperature. The solution was dialyzed against distilledwater for 3 days, neutralized by NaOH, and lyophilized to a white powder(32 mg, 107%). Further analysis of the recombinant chondroitin sulfatedemonstrated a molecular weight of 29 kDa, 5.2% of sulfur.

In another experiment, above-mentioned chondroitin (50 mg) wassolubilized into 1.0 mL of dry formamide (FA) at an ambient temperature.When the solution became clear, chlorosulfonic acid (5 eq. of CHdisaccharide unit) was slowly added and maintained with continuousstirring for 20 min. The sulfation reaction was stopped by addition of3× vol. of 1 M sodium acetate solution and was allowed to stand foranother 10 min. at room temperature. The solution was dialyzed againstdistilled water for 3 days, neutralized by NaOH, and lyophilized to givea white powder (47 mg, 94%). The analysis of the recombinant chondroitinsulfate showed a Mw of 33 kDa, 5.2% of sulfur.

Example 16

This example illustrates the chondroitin biosynthetic region 2 gene set(kfoABCFG) is sufficient for maximal enhancement of chondroitinproduction in E. coli strain MSC562.

A set of plasmids containing combinations of the regions 1, 2, and 3(R1, R2, and R3) gene sets was prepared from pBR1052 (FIG. 8K) and pDD67(FIG. 8J). As described above, sets of genes were deleted by digestionof a starting plasmid with particular restriction enzymes; generation ofblunt ends, for example, with T4 polymerase; and ligation of theresulting vector fragments. E. coli strain MSC188 (Example 3) wastransformed with the ligation reactions, and selected antibioticresistant transformants were evaluated for the desired features. Due tothe fact that pBR1052 contains a second Pm promoter preceding the region1 gene set, some of the plasmids described here also contain a second Pmpromoter. All of these described plasmids contain xylS. In Table 11below, R1=kpsFEDUCS, R2=kfoABCFG, and R3=kpsMT. Note that a “Pm:R2”plasmid was previously constructed (pCX039; Example 4). DNA maps forplasmids pCX096 (SEQ ID NO:149), pCX097, pCX100, pCX101 (SEQ ID NO:150),and pCX102 are shown in FIGS. 14A, 14B, 14C, 14D, and 14E, respectively.Plasmids pCX097 and pCX101 were used as starting plasmids for pCX100 andpCX102, respectively.

TABLE 11 retained starting plasmid kfo gene gene sets plasmid enzymesused name structure R1, R2 pBR1052 MluI + SbfI pCX096 Pm:R2/Pm:R1 R1, R3pBR1052 AvrII + NheI pCX097 Pm:R3/Pm:R1 R1 pCX097 PacI + SbfI pCX100Pm:R1 R2, R3 pDD67 PacI + PmeI pCX101 Pm:R3, R2 R3 pCX101 AvrII + NheIpCX102 Pm:R3

Each of the final plasmids was transformed into host strain MSC562resulting in the strains given in Table 12 below. These strains andpre-existing control strains were grown in shake flasks with 2×M9/tet5medium, induced with 1 mM meta-toluic acid at OD600 values ofapproximately 0.1-0.12, and evaluated for growth (OD600) and rCHproduction after 72 hours.

TABLE 12 rCH OD600 (μg/mL) strain plasmid properties 72 hr 72 hr MSC563pDD63 (empty) 5.25 107 MSC564 pCX039 Pm:R2 3.52 678 MSC681 pDD66 Pm:R3,R2, R1 7.46 522 MSC682 pDD67 Pm:R1, R3, R2 8.40 208 MSC683 pDD96Pm:R2/Pm:R1 6.96 729 MSC684 pCX097 Pm:R3/Pm:R1 2.32 172 MSC687 pCX100Pm:R1 5.83 292 MSC688 pCX101 Pm:R3, R2 7.80 277 MSC689 pCX102 Pm:R3 5.24121 MSC690 pBR1052 Pm:R3, R2, Pm: R1 6.29 140

The greatest productivities were seen in strains with plasmids carryingonly region 2 (MSC564) or the combination of regions 2 and 1 (MSC683).The combination of regions 2 and 3 (MSC688) resulted in lowerproductivity. In fact, the presence of plasmid-borne region 3 appearedinhibitory in other relevant strain comparisons as well (for example,MSC683 vs. MSC690). These findings support the approach of increasingrCH productivity by increasing just the region 2 copy number in strainMSC562.

Example 17

This example illustrates the positive copy number effect of chondroitinbiosynthetic gene region 2 requires all five of the kfoABCDG genes.

Plasmid pCX039 (FIG. 8Q; containing the region 2 genes kfoABCFG) drove alarge (8 to 10-fold) increase in rCH production when present in host E.coli strain MSC562 (as compared to MSC562 containing the empty vectorpDD63). Using methods described above (see Example 4) for the deletionof genes from pDD66 and pDD67, two sets of plasmids were derived frompCX039 to demonstrate the roles of individual region 2 genes onstimulation of rCH production by pCX039.

One set of five plasmids was designed to evaluate the effects of theremoval of one of the region 2 genes. In an E. coli host such as MSC562,one copy of these genes would still be present (integrated into thechromosome). This set of plasmids included pCX039 derivatives from whicheach of the kfoABCFG has been individually deleted. Table 13 below liststhe restriction enzymes used to delete the kfo genes and the names ofthe resulting plasmids. Example 4 above describes the derivation ofpCX044 in detail. All plasmids contain xylS.

TABLE 13 deleted gene enzyme used plasmid name kfo genes kfoA RsrIIpCX082 (SEQ ID NO:152) kfoBCFG kfoB BstBI pCX044 (SEQ ID NO:43) kfoACFGkfoC SpeI pCX075 (SEQ ID NO:153) kfoABFG kfoF AflII pCX081 (SEQ IDNO:151) kfoABCG kfoG NheI pCX092 (SEQ ID NO:154) kfoABCF

These plasmids were transformed into host stain MSC562 (chromosomalcopies of regions 1, 2 and 3 plus xlyS) to generate the strains shown inTable 14 below. Cultures were grown in 2×M9 medium (with 10 g/L glyceroland 2 μg/mL Tet) at 300, induced with 1 mM mTA at OD600 values ofapprox. 0.1, and assayed for rCH production after 72 hours of growth.

TABLE 14 rCH OD600 (μg/mL) Strain Plasmid Properties 72 hr 72 hr MSC563pDD63 (empty) 6.61 92 MSC564 pCX039 kfoABCFG 3.24 917 MSC566 pCX081kfoABCG (ΔkfoF) 6.88 455 MSC567 pCX082 kfoBCFG (ΔkfoA) 7.05 621 MSC640pCX044 kfoACFG (ΔkfoB) 5.81 520 MSC641 pCX075 kfoABFG (ΔkfoC) 6.87 712MSC643 pCX092 kfoABCF (ΔkfoG) 3.91 775

These results indicate that all five region 2 genes (kfoABCFG) arerequired to achieve maximal productivity under these conditions. Theresults for MSC563 also indicate that the expression of the kfoABCFGgenes from the chromosomal insertion is sufficient to supportsignificant ICH production in the absence of plasmid-borne gene copies.

A second set of pCX039 derivatives was designed to evaluate the effectsof the presence of individual plasmid-borne region 2 genes onenhancement of rCH titers in host strain MSC562. As a means of keepingexpression levels comparable among the resulting plasmids, thepromoter-proximal kfoA gene was retained in all constructs. While thisdesign strategy does not allow for evaluation of plasmid-encoded kfoB,C, F, or G genes in complete isolation, the retention of the kfoA geneand the resulting fixed relationship between the Pm promoter and thefirst reading frame in all of these plasmids are expected to makeexpression levels from the Pm promoter comparable. Using the samestrategies as described above, the following derivations (Table 15) werecarried out. All plasmids contain xylS.

TABLE 15 Retained Starting gene(s) plasmid Enzyme(s) used Plasmid namekfo genes pCX044 SpeI pCX050 kfoAFG (kfoACFG) kfoF (+kfoA) pCX050 NheIpCX070 kfoAF kfoA pCX070 AflII pCX095 kfoA kfoB (+kfoA) pCX039 NheI +SpeI pCX093 kfoAB (kfoABCFG) kfoC (+kfoA) pCX044 AflII pBR1077 kfoACGpBR1077 NheI pBR1082 kfoAC kfoG (+kfoA) pCX050 AflII pCX094 kfoAG

Each of the final set of plasmids was transformed into host strainMSC562 resulting in the strains given in Table 16 below. These strainsand MSC563 and MSC564 controls were grown in shake flasks with 2|M9/tet2medium, induced with 1 mM meta-toluic acid at OD600 values of 0.08-0.18,and evaluated for growth (OD600) and rCH production after 68 hours.

TABLE 16 OD600 rCH (μg/mL) strain plasmid properties 68 hr 68 hr MSC563pDD63 (empty) 4.82 103 MSC564 pCX039 kfoABCFG 7.91 861 MSC656 pCX095kfoA 5.06 146 MSC657 pCX093 kfoAB 5.18 236 MSC658 pBR1082 kfoAC 5.22 341MSC659 pCX070 kfoAF 4.99 198 MSC660 pCX094 kfoAG 5.53 130

These data demonstrate that none of the K4 region 2 genes (kfoABCFG)individually are sufficient to maximally stimulate rCH production in theMSC562 host strain. Taken together with the findings described above(e.g., Example 16), it is clear that including all five genes of theregion 2 gene set results in maximal enhancement of rCH production.

Example 18

This example illustrates constructs for increased chromosomal copynumber of chondroitin biosynthetic gene region 2 for greater chondroitinproduction.

Example 12 demonstrates that the addition of a single chromosomal copyof the region 2 gene set (kfoABCFG) in an E. coli host alreadycontaining single chromosomal copies of regions 1, 2, and 3 leads to asignificant 20-30% increase in rCH production. Example 11 demonstratesthat a similar host containing a multi-copy plasmid carrying the region2 gene set leads to a 2-300% increase in rCH production. With the goalof generating high-producing, plasmid-free strains, this exampledescribes the construction and use of plasmids designed to increase thechromosomal complement of region 2 copies (driven by the Pm promoter) byinserting them into various non-essential chromosomal genes chosenspecifically to facilitate identification of such insertions. It isnoted that in using the homology-driven “pop-in/pop-out” methodology(see Examples 4 and 12) to successively insert copies of region 2 genesets into different loci in the host E. coli chromosome, there isincreasing competition for undesired targeting (homology-drivenrecombination) into pre-existing region 2 inserts instead of the desiredlocus. Therefore, having a means of initially identifying strainscontaining inserts at the desired locus by simple colony screeninginstead of by more laborious and time-consuming PCR becomes increasinglybeneficial as the number of region 2 copies in the host strain rises.

In this example, three E. coli target loci are described. These are thegenes lacZ, mtlA, and the fruBKA operon (referred to as “fruA” forsimplicity in some cases) that are necessary for growth on the sugarslactose, mannitol, and fructose, respectively, but not for growth onother carbon sources such as glucose or glycerin. Colonies of strainsdisrupted for these genes can be visually identified on indicator agarssuch as MacConkey's (Miller, J H, Experiments in Molecular Genetics,1972) by colony color differences: pink/red for strains able to utilizethe incorporated sugar and white/light pink for strains with genedisruptions (e.g., insertions). Alternatively, LB/Xgal/IPTG agar medium(ibid.) can be used to detect defects in lactose metabolism: bluecolonies for strains able to utilize lactose and white/buff colonies forstrains unable to utilize lactose (such as strains with insertions intothe lacZ gene). Methods such as these, which use color differences,allow the visual identification of strains likely to have insertionsinto the desired locus among a population of colonies with insertionselsewhere. Those skilled in the art will recognize that other targetloci exist in E. coli that will allow the screening or selection ofdesired insert events; nonlimiting examples include pepP, pepQ,feuA(cirA), malB(lamB), nupA(tsx).

To facilitate the use of plasmid pMAK705 for insertion of region 2(“R2”) into the fruBKA, lacZ, and mtlA genes, a derivative of pMAK705containing a multi-cloning site was first developed. Primers DHD266c andDHD267c contain one-strand halves of a multiple restriction (cloning)site (NodI, XhoI, AscI, SalI, BglII, HindIII) and are complementaryexcept for 2-base single strand ends that (when annealed) are compatiblewith overhangs generated by digestion of pMAK705 with AseI and ClaI.Neither AseI nor ClaI restriction sites are regenerated upon ligation ofthese compatible ends.

DHD266c (SEQ ID NO: 155)TAGCGGCCGCATACTCGAGCATGGCGCGCCTAACGTCGACTAAGATCTCT AAGCTT DHD267c(SEQ ID NO: 156) CGAAGCTTAGAGATCTTAGTCGACGTTAGGCGCGCCATGCTCGAGTATGCGGCCGC

Plasmid pMAK705 was digested with AseI and CaI, and the vector fragmentwas gel-purified. Phosphorylated oligonucleotides DHD266c and DHD267cwere annealed (200 nM each oligonucleotide, 90° C. for 5 min, slowcooling to 50° C. for 30 min), and then ligated to the pMAK705 vectorfragment. Ligation reactions were transformed into E. coli NEB10β withselection for chloramphenicol resistance. Plasmids in isolatedtransformants were screened by PCR and then for the presence of MCSrestriction enzyme sites. Sequencing of the MCS region identified aplasmid with the desired structure. This plasmid was named pMAK705pl(SEQ ID NO:157; FIG. 14Q).

The construction of three vectors for insertion of R2 into the fruBKA,lacZ, and mtlA loci all took the same two-step approach. In a firststep, the upstream and downstream regions of homology were generated foreach target locus using PCR primers that allow annealing between the“inside” ends of the PCR products. This region of homology encompassesmultiple restriction sites that were later used for addition of R2. In asecond step, the upstream and downstream PCR products for each locus aremixed in a PCR reaction with the two “outside” primers used originallyto synthesize the individual template members. Due to the end homologiesdesigned into the upstream and downstream PCR products (now templates instep 2), the results of the step 2 reactions were DNA fragmentscomprising the appropriate orientations of the upstream and downstreamregions flanking multi-cloning sites. The PCR products from step 2 weredigested with enzymes whose recognition sequences were designed into the“outside” primers: NotI for upstream ends and HindIII for the downstreamends. These fragments were then individually cloned into pMAK705pl (SEQID NO:157) digested with NotI and HindIII. The three resulting plasmidscontained approximately 900-1000 bp of properly oriented upstream (UP)and downstream (DN) regions flanking multi-cloning sites (MCS) to beused to accept R2 copies. For pBR1093, the MCS replaced about 20 bp oflacZ coding region. For pBR1094, the MCS was inserted into the mtlAcoding region. For pBR1095, the MCS replaced the 3-prime end of fruB,all of fruK, and the 5-prime end of fruA. The primers used to preparethese intermediate constructs are listed in Table 25 below.

To prepare the region 2 gene set for cloning into pBR1093, pBR1094, andpBR1095, the kfoABCFG genes were excised (without the Pm promoter) frompCX074 (see Example 11) using PacI+ClaI. The purified R2 fragment wasthen cloned into pJ201:11352 (see FIG. 8B) digested with the sameenzymes. This resulted in plasmid pBR1096 in which the kfoABCFG geneswere again orientated behind the Pm promoter. Now, however, Pm:R2 couldbe isolated from pBR1096 as a XhoI/AscI fragment for cloning intopBR1093 (FIG. 14S), pBR1094 (FIG. 14V), and pBR1095 (FIG. 14W). Table 17provides the designation of the final replacement pMAK705-based Pm:R2insertion plasmids pBR1100 (for the lacA locus), pBR1101 (for the mtlAlocus), and pBR1102 (for the fruBKA locus). The sequences for primersDHD280c, DHD281c, DHD283, DHD285, DHD268c, DHD269c, DHD271, DHD273,DHD274c, DHD275c, DHD277, and DHD279 are shown in SEQ ID NOs:158-169,respectively.

TABLE 17 Step 1 PCRs UP/MCS/DN final plasmid locus upstream downstreamStep 2 PCR plasmid UP/Pm:R2/DN fruBKA DHD280c x DHD283 x DHD280c xpBR1095 pBR1102 DHD281c DHD285 DHD285 (SEQ ID NO: 170) lacZ DHD268c xDHD271 x DHD268c x pBR1093 pBR1100 DHD269c DHD273 DHD273 (SEQ ID NO: 171) mtlA DHD274c x DHD277 x DHD274c x pBR1094 pBR1101 DHD275c DHD279DHD279 (SEQ ID NO: 172)

The previously-described “pop-in/pop-out” methodology was used withplasmids pBR1100 (SEQ ID NO: 171; FIG. 14T). pBR1101 (SEQ ID NO:172;FIG. 14V) and pBR1102 (SEQ ID NO:170; FIG. 14X) to impart additionalPm:R2 copies to chosen E. coli strains. Strains were transformed withpBR1100, pBR1101, or pBR1102 at 30° C. with selection forchloramphenicol resistance. Transformants were then plated at 43° C. toMacConkey/fructose/Cm agar (for pBR1102 transformants),MacConkey/mannitol/Cm agar (for pBR1101 transformants), orLB/Xgal/IPTG/Cm agar (for pBR1100 transformants). Colonies that wereconspicuously less colored were chosen for further analysis. Amongthese, strains with plasmids integrated into the target loci wereidentified by PCR. Strains with successfully-integrated plasmids werethen grown for multiple (e.g., 20-30) generations in the absence ofchloramphenicol selection. Colonies derived from these cultures werescreened for chloramphenicol sensitivity (reflecting excision of theplasmids) and defects in sugar metabolism (reflecting retention of thetargeted Pm:R2 insertion). Isolates with the desired phenotypes wereevaluated by PCR for the correct chromosomal structures. FIG. 15diagrams the multiple steps in strain derivation utilizing the methodsdescribed in this and other Examples. As an illustration and summary,strain MSC702 contains the following key elements:Pm[kpsMTkfoABCFG]Pm[kpsFEDUCS] inserted at the colanic acid locus,Psyn[xylS] inserted at the fhuA locus, Pm[kfoABCFG] inserted at thefruBKA, lacZ and mtlA loci, and (presumed due to its derivation fromMSC691; see Example 19) 8 base pair changes within the leuB gene.

Example 19

This example illustrates the identification and correction ofspontaneously-occurring auxotrophy in E. coli strains.

During the course of evaluating recombinant E. coli strains for rCHproduction in minimal growth medium, it was discovered that certainstrains did not grow. It was subsequently determined that strain MSC561grew on minimal medium only when amended with a source of leucine; i.e.,this strain is a leucine auxotroph. Sequencing of the leucinebiosynthetic operon leuABCD in MSC561 revealed that the strain hadspontaneously acquired a single base pair deletion in the leuB genecoding region (a C/G base pair at position 383 of the coding region)during its derivation from the leucine prototroph MSC467 (see Example10). This deletion results in a reading frame shift and pre-maturetranslation termination. This defect was not initially detected becausegenetic manipulations and early production tests were conducted incomplex media. It is very unlikely that this mutation was the result ofprevious targeted recombination at the fhuA and colanic acid locibecause they are not closely linked to leuB (fhuA and leuB are about 85Kb apart; the colanic acid operon and leuB are about 2 Mb apart). Allstrains immediately or sequentially derived from MSC561 by addition ofR2 copies (such as MSC627, MSC650, MSC646, MSC679, and MSC700; see FIG.15) were also leucine auxotrophs and contained the identical deletion inthe leuB sequence. Strains in a separate lineage of MSC467 (MSC537,MSC562, and MSC619) are leucine prototrophs. (see FIG. 15).

Two approaches were used to convert selected leucine auxotrophs toprototrophs (therefore allowing growth in minimal media without addedleucine). In one approach, large numbers (about 10⁶-10⁷) of cells of anauxotrophic strain were applied to a minimal medium agar plate followedby incubation at 30° C. for 3-7 days. Typically, several coloniesdevelop under these conditions. Strains isolated from these colonies(“spontaneous revertants”) reproducibly grow on solid and liquid minimalmedia without leucine. Sequence analysis of the leuB gene of selectedrevertants revealed (in most cases) small insertions or deletions nearthe site of the original single base pair deletion that result in therestoration of the correct leuB reading frame. Table 18 below providesthe locations of the nucleotide changes with coordinates relative to theleuB coding region. The LeuB enzymes in these spontaneous revertantstrains have altered amino acid sequences in this region, but thechanges from the native structure appear to allow sufficient function tosupport growth in leucine-free media. In spontaneous revertant MSC692,no compensating nucleotide changes in leuB were detected. The nature ofthe genetic change in this strain is uncharacterized.

In a second approach toward converting leucine auxotrophs toprototrophs, the spontaneous mutation in leuB of selected strains wasspecifically corrected to native sequence. PCR primers BLR513 (SEQ IDNO:173) and BLR516 (SEQ ID NO:174) were used to amplify a 646 base pairregion of the native leuB gene using gDNA from wild type E. coli W3110as template. The site of the base pair found deleted in MSC561 was 288bp from the upstream end of this PCR fragment, and the PCR primersgenerate HindIII and XhoI ends for cloning into pMAK705pl (Example 18;SEQ ID NO:157; FIG. 14Q) resulting in pBR1103 (SEQ ID NO:175; FIG. 14R).The leuB gene fragment in pBR1103 extends from the HindIII restrictionsite at bp=5059-5064 of SEQ ID NO:175 to the XhoI restriction site atbp=5712-5717 of SEQ ID NO: 175. These restriction sites are not part ofthe natural leuB sequence but were introduced for cloning purposes byPCR with primers BLR513 and BLR516.

Standard “pop-in/pop-out” methodology was then used to replace thedefective leuB region with the native region in auxotrophic strainsMSC650, MSC679, and MSC700 to give prototrophic strains MSC722. MSC723,and MSC724, respectively. Briefly, initial transformants of MSC650,MSC679, and MSC700 with pBR1103 were selected on LB/Cm34 plates at 30°C. Selected transformants were plated to LB/Cm34 at 43° C., and isolatedsurvivors were confirmed by PCR to have pBR1103 integrated at the leuBlocus. Selected integrants were grown in LB (no Cm) at 30° C. for about10 generations, then in 2×M9 medium (no Cm, no leucine) for about 15generations. Strains derived from colonies on LB plates isolated fromthese cultures were screened for chloramphenicol-sensitivity andprototrophy. DNA sequencing of the leuB gene in prototrophic,chloramphenicol-sensitive strains derived from each of the three initialparent strains confirmed that the original wild type sequence wasrestored. FIG. 15 shows the derivation of these strains in relation toother strains described herein.

TABLE 18 leucine deletion (Δ) or inser-  net  pheno- tion (Ω) in leuB; bp strain type (leuB coordinates) change W3110 + none na MSC651 −ΔC(383) −1 MSC650 − ΔC(383) −1 MSC669 + ΔC(383), ΔGG (345-346) −3MSC670 + ΔC(383), ΔCTGTCCGCTGCGTG −15 (360-373) MSC671 +ΔC(383), ΩCGCAAACGGC  +9 (at 394) MSC675 + ΔC(383), ΩT (at 340) 0MSC677 + ΔC(383), ΩAACT (at 339) +3 MSC678 + ΔC(383), ΩG (at 345) 0MSC691 + ΔC(383), ΩCATCCTG +6 (at 410) MSC692* + ΔC(383) −1 MSC693 +ΔC(383), ΔCA (374-375) −3 MSC694 + ΔC(383), ΩA (at 387) 0 MSC722 + none0 MSC723 + none 0 MSC724 + none 0 *No compensating changes in the leuBregion were detected from about 200 bp upstream of the leuB start codon(i.e., the 3-prime end of leuA) to about 200 bp upstream of the leuBstop codon (i.e., leuB coordinates 1 to approx. 850 of the 1089 totalbase pairs of the leuB coding region).

rCH production in strains MSC722, MSC723, and MSC724 (thespecifically-corrected Leu+prototrophs) was compared to production instrains with identical K4 gene complements and arrangements but derivedby spontaneous conversion to prototrophy: MSC677, MSC692, and MSC702,respectively (see FIG. 15). The six strains were grown in duplicate 2×M9flasks at 30° C. and induced with 1 mM mTA at OD600 values ofapproximately 0.1. Samples of culture broths at 71 hours post-inductionwere assayed for rCH content as described above. The average OD600 andrCH concentrations are shown in Table 19.

TABLE 19 OD600 at 71 hr rCH (μg/mL) strain Leu+ (each flask); average(each flask); average MSC677 spontaneous (7.63, 6.62); 7.12 (362, 478);420 MSC722 specific correction (5.46, 5.20); 5.33 (226, 230); 228 MSC692spontaneous (6.45, 8.50); 7.48 (290, 339); 315 MSC723 specificcorrection (7.90, 6.88); 7.39 (450, 400); 425 MSC702 spontaneous (6.20,6.40); 6.30 (344, 328); 336 MSC724 specific correction (6.04, 6.75);6.40 (415, 428); 421

In two of the three strain pairs, rCH production is greater in thestrain with the specific correction to leuB. but final cell density (asmeasured by terminal OD600) was similar. These results demonstrate thebenefit in terms of rCH production of specifically correcting thespontaneous mutation discovered during the course of E. coli straindevelopment.

Example 20

This example describes recombinant DNA-mediated production ofchondroitin in E. coli in fermentors.

1. 10-L Fermentation of E. coli (MSC537)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC537 was cultivated using glycerine as acarbon source. The fermentor was batched with the following medium(Table 20A) to a volume of 6 liters using de-ionized water.

TABLE 20A Medium Component Amount NZ Amine HD 320.0 g Tastone 154  8.0 gKH₂PO₄  80.0 g MgSO₄•7H₂O  8.0 g Na₂SO₄  48.0 g Sodium citrate  2.4 gDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 201) were added aseptically:

TABLE 20B Component Amount Glycerine (heat-sterilized) 32.0 g ThiamineHCl (filter-sterilized) 320 μg Trace Metal solution (filter-sterilized,recipe below) 24 mL Trace Metal Solution Component ConcentrationFeCl₃•6H₂O  27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/L Na₂MoO₄•6H₂O 2.0 g/LCaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/L HCl, concentrated160 mL/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4.25 hours, then cultivated for 69 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution) duringcultivation. After 69 hours, the fermentor was autoclaved and harvestedby centrifugation. The fermentation control conditions and product yieldare shown in Tables 20C and 20D, respectively.

TABLE 20C Fermentation Control Conditions Temperature 29.6-30.1° C. pH7.08-7.30 Agitation 150-450 cps (cm per sec) Dissolved oxygen 50%(setpoint) Airflow 4.0-6.4 L/min Oxygen flow    0-1 L/min Glycerine 0-6g/L (in-tank concentration) Inoculum 58 mL from shake flask, OD₆₀₀ = 6.9

TABLE 20D Product Yield Chondroitin yield 4.50 g/L Glycerine consumed 185 g/L Final OD₆₀₀ 912. 2-L Fermentation of E. coli (MSC564)

Using a 2-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC564 was cultivated using glycerine as acarbon source. The fermentor was batched with the following medium(Table 21A) to a volume of 1.5 liters using de-ionized water:

TABLE 21A Medium Component Amount NZ Amine HD 32.0 g  Tastone 154 0.8 gKH₂PO₄ 8.0 g MgSO₄•7H₂O 0.8 g Na₂SO₄ 4.8 g Sodium citrate 0.48 g  Dow1520US antifoam 0.16 mL De-ionized water As needed to reach 1.5 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 21B) were added aseptically:

TABLE 21B Component Amount Glycerine (heat-sterilized) 6.4 g ThiamineHCl (filter-sterilized) 64 μg Tetracycline 8.0 mg Trace Metal solution(filter-sterilized, recipe below) 4.8 mL Trace Metal Solution ComponentConcentration FeCl₃•6H₂O  27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LHCl, concentrated 160 mL/L

The fermentor was inoculated with a typical seed culture, thencultivated for 66 hours and fed 170 mL of a carbon feed (consisting of a625 g/L glycerine solution) during cultivation. After 5 hours ofcultivation, it was induced with 2 mM m-TA. The fermentation wascontrolled for pH, dissolved oxygen, temperature, glycerineconcentration, and acetate concentration (byproduct of carbonmetabolism). The glycerine feed rate was adjusted based on the acetateconcentration with a target of <2 g/L acetate. The target glycerineconcentration was <5 g/L. The fermentation was run for 66 hours, atwhich point, glycerine consumption declined to <1.5 g/L/hour. Due tosampling and evaporation, the final volume was 1.35 L. The controlconditions and product yield are listed below. After 66 hours, thefermentor was autoclaved and harvested by centrifugation. The recoveredvolume after centrifugation was approximately 1 liter of supernatant.The fermentation control conditions and product yield are shown below inTables 21C and 21D, respectively.

TABLE 21C Fermentation Control Conditions Temperature 29.8-30.1° C. pH7.1-7.30 Agitation 150-275 cps Dissolved oxygen 50% (setpoint) Airflow0.7-1.2 L/min Oxygen flow 0.1-0.6 L/min Glycerine 0-12 g/L (in-tankconcentration) Inoculum 8 mL from shake flask, OD₆₀₀ 10.2 (17.5 hourflask)

TABLE 21D Product Yield Chondroitin yield 6.2 g/L Glycerine consumed 79g/L (1.35 L final vol.) Final OD₆₀₀ 22.3

The next three fermentations (3-5) were taken from one experiment wherethree 10 L reactors were run side-by-side under the same conditionscomparing strains MSC619, MSC677 and MSC702 containing different numbersand arrangements of the region 2 gene set in the host chromosome (seeExample 19: FIG. 15). Briefly, MSC619 and MSC677 each have three totalcopies of region 2, but one copy in MSC619 is driven by the Psynpromoter instead of the Pm promoter. Strain MSC702 has four copies ofthe region 2 gene set, all driven by Pm.

3. 10-L Fermentation of E. coli (MSC619)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC619 was cultivated using glycerine, caseinhydrolysate and ammonium hydroxide as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 22A) to a volume of 6 liters using de-ionized water:

TABLE 22A Medium Component Amount NZ Amine HD 160.0 g  Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g  Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 22B) were added aseptically:

TABLE 22B Component Amount Glycerine (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 μg Trace Metal solution(filter-sterilized, recipe below) 24 mL Trace Metal Solution ComponentConcentration FeCl₃•6H₂O  27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid  33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4.2 hours, then cultivated for 80 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution) and a nitrogenfeed in the form of 4N ammonium hydroxide. The carbon and nitrogen feedswere adjusted manually based on sample readings taken offline on theNOVA 300A Bioanalyzer. The concentration of glycerine was targeted tomaintain less than 1 g/L and the feedrate was reduced further in thepresence of acetate accumulation. The ammonia concentration target was100 mg/L or less of ammonia in the broth. Acid in the form of 2Nsulfuric acid and base in the form of 4N sodium hydroxide were addedautomatically during cultivation to control pH. Antifoam was also fedautomatically to the fermentor to control foaming of the broth. Thefermentation control conditions and product yield are shown below inTables 22C and 22D, respectively.

TABLE 22C Fermentation Control Conditions Temperature 29.9-30.1° C. pH7.2-7.3 Agitation 150-360 cps Dissolved oxygen 50% (setpoint) Airflow5.3-6.4 L/min Oxygen flow   0-1.1 L/min Glycerine 0-9.1 g/L (in-tankconcentration) Inoculum 50 mL from shake flask, OD₆₀₀ = 7.2

Table 22D Product Yield Chondroitin yield 3.45 g/L (7.3 L final volume)Glycerine consumed 135 g/L Final OD₆₀₀ 644. 10-L Fermentation of E. coli (MSC677)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC677 was cultivated using glycerine, caseinhydrolysate and ammonium hydroxide as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 23A) to a volume of 6 liters using de-ionized water.

TABLE 23A Medium Component Amount NZ Amine HD 160.0 g Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 23B) were added aseptically:

TABLE 23B Component Amount Glycerine (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 μg Trace Metal solution 24mL (filter-sterilized, recipe below) Trace Metal Solution ComponentConcentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4.2 hours, then cultivated for 80 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of 4N ammonium hydroxide. The carbon and nitrogen feedswere adjusted manually based on sample readings taken offline on theNOVA 300A Bioanalyzer. The concentration of glycerine was targeted tomaintain less than 1 g/L and the feed rate was reduced further in thepresence of acetate accumulation. The ammonia concentration target was100 mg/L or less of ammonia in the broth. Acid in the form of 2Nsulfuric acid and base in the form of 4N sodium hydroxide were addedautomatically during cultivation to control pH. Antifoam was also fedautomatically to the fermentor to control foaming of the broth. Thefermentation control conditions and product yield are shown in Tables23C and 23D, respectively.

Table 23C Fermentation Control Conditions Temperature 29.7-30.2° C. pH6.98-7.37 Agitation 150-440 cps Dissolved oxygen 50% (setpoint) Airflow5.0-6.4 L/min Oxygen flow   0-1.9 L/min Glycerine 0-7.5 g/L (in-tankconcentration) Inoculum 50 m/L from shake flask, OD₆₀₀ = 6.7

TABLE 23D Product Yield Chondroitin yield 4.3 g/L (8.4 L final volume)Glycerine consumed 143 g/L Final OD₆₀₀ 575. 10-L Fermentation of E. coli (MSC702)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC702 was cultivated using glycerine, caseinhydrolysate and ammonium hydroxide as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 24A) to a volume of 6 liters using de-ionized water.

TABLE 24A Medium Component Amount NZ Amine HD 160.0 g Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 24B) were added aseptically.

TABLE 24B Component Amount Glycerine (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 μg Trace Metal solution(filter-sterilized, recipe low) 24 mL Trace Metal Solution ComponentConcentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4.2 hours, then cultivated for 80 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of 4N ammonium hydroxide. The carbon and nitrogen feedswere adjusted manually based on sample readings taken offline on theNOVA 300A Bioanalyzer. The concentration of glycerine was targeted tomaintain less than 1 g/L and the feed rate was reduced further in thepresence of acetate accumulation. The ammonia concentration target was100 mg/L or less of ammonia in the broth. Acid in the form of 2Nsulfuric acid and base in the form of 4N sodium hydroxide were addedautomatically during cultivation to control pH. Antifoam was also fedautomatically to the fermentor to control foaming of the broth. Thefermentation control conditions and product yield are shown in Tables24C and 24D, respectively.

TABLE 24C Fermentation Control Conditions Temperature 29.8-30.2° C. pH6.8-7.3 Agitation 150-490 cps Dissolved oxygen 50% (setpoint) Airflow4.0-6.4 L/min Oxygen flow   0-2.4 L/min Glycerine 0-7.0 g/L (in-tankconcentration) Inoculum 50 mL from shake flask, OD₆₀₀ = 6.7

TABLE 24D Product Yield Chondroitin yield 5.3 g/L (8.9 L final volume)Glycerine consumed 151 g/L Final OD₆₀₀ 60

To summarize experiments 3-5, strains MSC619, MSC677, and MSC702 yielded3.45, 4.3 and 5.3 g/L chondroitin, respectively, demonstrating theeffects of region 2 arrangement (context) and copy number in enhancingchondroitin productivities.

6. 10-L Fermentation of E. coli (MSC702)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC702 was cultivated using glycerine, caseinhydrolysate and ammonium hydroxide as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 25A) to a volume of 6 liters using de-ionized water.

Table 25A Medium Component Amount NZ Amine HD 160.0 g Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 25B) were added aseptically.

TABLE 25B Component Amount Glycerine (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 μg Trace Metal solution 24mL (filter-sterilized, recipe below) Trace Metal Solution ComponentConcentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4 hours, then cultivated for 82 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of 4N ammonium hydroxide. The carbon and nitrogen feedswere adjusted manually based on sample readings taken offline on theNOVA 300A Bioanalyzer. The concentration of glycerine was targeted tomaintain less than 1 g/L and the feed rate was reduced further in thepresence of acetate accumulation. The ammonia concentration target was100 mg/L or less of ammonia in the broth. Acid in the form of 2Nsulfuric acid and base in the form of 4N sodium hydroxide were addedautomatically during cultivation to control pH. Antifoam was also fedautomatically to the fermentor to control foaming of the broth. Thefermentation control conditions and product yield are shown in Tables25C and 25D, respectively.

TABLE 25C Fermentation Control Conditions Temperature 29.8-32° C. pH7.1-7.3 Agitation 150-480 cps Dissolved oxygen 50% (setpoint) Airflow3.4-6.4 L/min Oxygen flow 0-3.1 L/min Glycerine 0-7.7 g/L (in-tankconcentration) Inoculum 50 mL from shake flask, OD₆₀₀ = 9.0

TABLE 25D Product Yield Chondroitin yield 8.3 g/L (8.4 L final volume)Glycerine consumed 123 g/L Final OD₆₀₀ 69

This run was designed as a repetition of fermentation experiment 5(above) but achieved a significant increase in chondroitin yield. Thedifference in yield is at least partly believed to be due to excessantifoam used in experiment 5 along with an increased level of acetatebuild up, both of which are considered to negatively affect chondroitinyield.

7. 50-L Fermentation of E. coli (MSC702)

Using a 50-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC702 was cultivated in a defined mediumusing glycerine, and ammonium hydroxide as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 26A) to a volume of 40 liters using de-ionized water.

TABLE 26A Medium Component Amount NaCl 50.0 g NH4Cl 100.0 g MgSO4•7H2O12.0 g CaCl2*2H2O 1.85 g Dow 1520US antifoam 5.0 mL De-ionized water Asneeded to reach 40.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 26B) were added aseptically.

TABLE 26B Component Amount Glycerine (heat-sterilized) 300 g Na2HPO4 240g KH₂PO₄ 120 g Thiamine HCl 2 mg (filter-sterilized) Trace Metalsolution 150 mL (filter-sterilized, recipe below) Trace Metal SolutionComponent Concentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0g/L Na₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5g/L Citric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4 hours, then cultivated for 91 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of 6N ammonium hydroxide. The carbon and nitrogen feedswere adjusted manually based on sample readings taken offline on theNOVA 300A Bioanalyzer. The concentration of glycerine was targeted tomaintain less than 1 g/L and the feed rate was reduced further in thepresence of acetate accumulation. The ammonia concentration target was100 mg/L or less of ammonia in the broth. Acid in the form of 3Nsulfuric acid and base in the form of 4N sodium hydroxide were addedautomatically during cultivation to control pH. Antifoam was fedmanually to the fermentor to control foaming of the broth. Thefermentation control conditions and product yield are shown in Tables26C and 26D, respectively.

TABLE 26C Fermentation Control Conditions Temperature 30° C. setpoint pH7.2 setpoint Agitation 150-375 cps Dissolved oxygen 50% setpoint Airflow30-40 L/min Oxygen flow  0-10 L/min Glycerine 0-7.8 g/L (in-tankconcentration) Inoculum 1000 mL from 2L fermentor, OD₆₀₀ = 6.9

TABLE 26D Product Yield Chondroitin yield 4.7 g/L (52 L final volume)Glycerine consumed 143 g/L Final OD₆₀₀ 114

This fermentation experiment demonstrates that high chondroitinproduction was achieved in defined (minimal) growth medium atintermediate fermentation scale.

The next two fermentations (8-9) were taken from one experiment wheretwo 10 L reactors were run side by side under the same conditionscomparing strains MSC702 and MSC724.

8. 10-L Fermentation of E. coli (MSC702)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC702 was cultivated using glycerine, caseinhydrolysate and ammonium sulfate as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 27A) to a volume of 6 liters using de-ionized water.

TABLE 27A Component Amount NZ Amine HD 160.0 g Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US 0.8 mL antifoam De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 27B) were added aseptically.

TABLE 27B Component Amount Glycerinr (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 g Trace Metal solution 24 mL(filter-sterilized, recipe below) Trace Metal Solution ComponentConcentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4 hours, then cultivated for 92 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of ammonium sulfate. The carbon and nitrogen feeds wereadjusted manually based on sample readings taken offline on the NOVA300A Bioanalyzer. The concentration of glycerine in the broth wastargeted to maintain less than 1 g/L and the feed rate was reducedfurther in the presence of acetate accumulation. The ammoniaconcentration target was 100 mg/L or less of ammonia in the broth. Acidin the form of 2N sulfuric acid and base in the form of 4N sodiumhydroxide were added automatically during cultivation to control pH.Antifoam was also fed automatically to the fermentor to control foamingof the broth. The fermentation control conditions and product yield areshown in Tables 27C and 27D, respectively.

TABLE 27C Fermentation Control Conditions Temperature 29.9-30.1° C. PH7.2-7.3 Agitation 150-460 cps Dissolved oxygen 50% (setpoint) Airflow5.0-6.4 L/min Oxygen flow   0-1.4 L/min Glycerine 0.2-11.4 g/L (in-tankconcentration) Inoculum 100 mL from shake flask, OD₆₀₀ = 1.7

TABLE 27D Product Yield Chondroitin yield 7.9 g/L (7.8 L final volume)Glycerine consumed 128 g/L Final OD₆₀₀ 509) 10-L Fermentation of E. coli (MSC724)

Using a 10-liter fermentor under typical fermentation conditions, aculture of E. coli strain MSC724 was cultivated using glycerine, caseinhydrolysate and ammonium sulfate as the main carbon and nitrogensources, respectively. The fermentor was batched with the followingmedium (Table 28A) to a volume of 6 liters using de-ionized water.

TABLE 28A Component Amount NZ Amine HD 160.0 g Tastone 154 4.0 gMgSO₄•7H₂O 8.0 g Na₂SO₄ 24.0 g Sodium citrate 2.4 g CaCl2*2H2O 296 mgDow 1520US antifoam 0.8 mL De-ionized water As needed to reach 6.0 L

After autoclaving of the fermentor containing the above medium, thefollowing components (Table 28B) were added aseptically.

TABLE 28B Component Amount Glycerine (heat-sterilized) 48.0 g KH₂PO₄48.0 g Thiamine HCl (filter-sterilized) 320 μg Trace Metal solution 24mL (filter-sterilized, recipe below) Trace Metal Solution ComponentConcentration FeCl₃•6H₂O 27 g/L ZnCl₂ 1.3 g/L CoCl₂•6H₂O 2.0 g/LNa₂MoO₄•6H₂O 2.0 g/L CaCl₂•2H₂O 2.5 g/L MnCl₂•4H₂O 3.3 g/L H₃BO₃ 0.5 g/LCitric acid 33 g/L

The fermentor was inoculated with a typical seed culture and inducedwith 2 mM m-TA after 4 hours, then cultivated for 92 hours and fed acarbon feed (consisting of a 625 g/L glycerine solution), and a nitrogenfeed in the form of ammonium sulfate. The carbon and nitrogen feeds wereadjusted manually based on sample readings taken offline on the NOVA300A Bioanalyzer. The concentration of glycerine in the broth wastargeted to maintain less than 1 g/L and the feed rate was reducedfurther in the presence of acetate accumulation. The ammoniaconcentration target was 100 mg/L or less of ammonia in the broth. Acidin the form of 2N sulfuric acid and base in the form of 4N sodiumhydroxide were added automatically during cultivation to control pH.Antifoam was also fed automatically to the fermentor to control foamingof the broth. The fermentation control conditions and product yield areshown in Tables 28C and 28D, respectively.

TABLE 28C Fermentation Control Conditions Temperature 29.8-30.2° C. pH7.2-7.3 Agitation 150-480 cps Dissolved oxygen 50% (setpoint) Airflow5.0-6.4 L/min Oxygen flow   0-1.4 L/min Glycerine 0.2-9.8 g/L (in-tankconcentration) Inoculum 50 mL from shake flask OD₆₀₀ = 7.7

TABLE 28C Product Yield Chondroitin yield 9.3 g/L (8.0 L final volume)Glycerine consumed 122 g/L Final OD₆₀₀ 47

Fermentations 8 and 9 in this Example demonstrate an improved rCH yieldof strain MSC724 over MSC702 in complex medium. This may be the resultof greater metabolic efficiency of the native LeuB enzyme in MSC724compared to the functional, but altered LeuB enzyme, in MSC702 (seeExample 19).

Example 21 This Example Describes the Recombinant DNA-MediatedProduction of Chondroitin in X. Campestris in Fermentors

10-L Fermentation of X. campestris (MSC480)

Using a 10-liter fermentor under typical fermentation conditions aculture of Xanthomonas campestris strain MSC480 was cultivated usingglucose as a carbon source. The fermentor was batched with the followingmedium (Table 29A) to a volume of 7.5 liters using de-ionized water:

TABLE 29A Medium Component Amount Malt Extract 200.0 g Tastone 154 80.0g KH₂PO₄ 17.0 g MgSO₄•7H₂O 5.0 g Na₂SO₄ 1.0 g CaCl₂•2H₂O 0.5 g Citricacid 20.0 g H₃BO₃ 60 mg ZnCl₂ 100 mg FeCl₃•6H₂O 200 mg Dow 1520US 2 mLantifoam De-ionized water As needed to reach 7.5 L

After autoclaving of the fermentor containing the above medium, 60 g ofglucose (heat-sterilized) was added aseptically.

The fermentor was inoculated with a typical seed culture, thencultivated for 70 hours and fed a carbon feed (consisting of an 871 g/Lglucose solution) during cultivation. After 70 hours, the fermentor wasautoclaved and harvested by centrifugation. The fermentation controlconditions and product yield are shown in Tables 29B and 29C,respectively.

TABLE 29B Fermentation Control Conditions Temperature 29.9-30.1° C. PH6.70-7.11 Agitation 150-250 cps Dissolved oxygen 20% (setpoint) Airflow5.0-8.0 L/min Glucose 13-27 g/L (in-tank concentration) Inoculum 850 mLfrom 2-L fermentor, OD₆₀₀ = 5.9

TABLE 29C Product Yield Chondroitin yield 1.86 g/L Glucose consumed 19g/L Final OD₆₀₀ 18

Example 22

This example illustrates an improved E. coli growth medium.

Examples 4, 7, and 8 above describe the use of complex TB medium forgrowth of rCH-producing recombinant E. coli K-12 strains. As normallyformulated, TB medium contains 5 g/L glycerine as the primary carbonsource. This example describes modifications to TB medium that enhancerCH volumetric and specific productivities in shake flasks.

A small culture of strain MSC564 was developed in TB/Tc5 medium at 30°C. for use as inocula. Standard TB medium (Sambrook et al., 1989; Difco“Terrific Broth”) was modified with 0.1 M MOPS buffer(4-morpholinepropanesulfonic acid; prepared from a 1.0M stock solutionpH-adjusted to 7.2 with NaOH), 10 vg/L glycerine (2× normal TB recipe),or both. Erlenmeyer shake flasks (250 mL) containing 50 mL of eachmedium were inoculated with the MSC564 culture to achieve OD600=0.03.The flasks were shaken (225 rpm) at 30° C. until OD600 values reachedapprox. 0.125, at which time meta-toluic acid was added to 1 mM toinduce rCH production. After 72 hours of continued shaking, pH and OD600measurements were taken, and 5 mL aliquots were autoclaved for 5-7 min.,cooled and stored frozen. rCH contents were determined as described inExample 14. The final OD600 and rCH concentrations are shown in Table30.

TABLE 30 medium final pH final OD600 rCH (μg/mL) TB 8.3 13.4 1017TB/0.1M MOPS pH 7.2 8.1 8.53 1244 TB/2x glycerine (10g/L) 6.0 9.64 676TB/MOPS/glycerine 7.4 12.2 1592

TB medium buffered and amended with extra glycerine (2× normal) resultedin a >50% increase in rCH titer (greater volumetric productivity)without additional cell density (greater specific productivity). Growthand productivity in medium with 2× glycerine but without buffer resultedin poor productivity, likely due to excess acid production fromglycerine. This demonstrates increased production capacity inrecombinant strains and provides higher productivity growth conditionsunder which to evaluate new E. coli strains.

The foregoing description of the present invention has been presentedfor purposes of illustration and description. Furthermore, thedescription is not intended to limit the invention to the form disclosedherein.

All of the various aspects, embodiments, and options described hereincan be combined in any and all variations.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1-64. (canceled)
 65. A construct comprising a kfoA, kfoC, and kfoF gene,wherein the construct does not contain a functional gene of one or moreof kfoD, orf3(kfoI), kfoE, or orf1(kfoH), and wherein the construct issuitable for producing chondroitin in a non-pathogenic bacterial hostcell.
 66. The construct of claim 65, wherein the construct furthercomprises a kfoG gene, a kfoB gene, or a combination thereof.
 67. Theconstruct of claim 65, wherein the chondroitin is non-fructosylated. 68.The construct of claim 65, wherein the construct further comprises akpsF, kpsE, kpsD, kpsU, kpsC, and kpsS gene.
 69. The construct of claim68, wherein the construct further comprises a kpsM and kpsT gene. 70.The construct of claim 68, wherein the chondroitin is secreted from thehost cell.
 71. The construct of claim 65, wherein the construct alsodoes not contain a functional gene of one or more of kpsM, kpsT, kpsE,kpsD, kpsC, or kpsS.
 72. The construct of claim 71, wherein thechondroitin is not secreted from the host cell.
 73. The construct ofclaim 65, wherein one or more genes are modified for optimal codon usagein a bacterial host cell.
 74. The construct of claim 65, comprising a K4gene cluster.
 75. A non-pathogenic bacterial host cell comprising theconstruct of claim
 65. 76. The non-pathogenic bacterial host cell ofclaim 75, wherein the non-pathogenic bacterial host cell is or isderived from a non-pathogenic organism selected from the groupconsisting of Escherichia, Pseudomonas, Xanthomonas, Methylomonas,Acinetobacter, and Sphingomonas.
 77. The non-pathogenic bacterial hostcell of claim 76, wherein the bacterial host cell is a bacterial strainselected from the group consisting of MSC279, MSC280, MSC315, MSC316,MSC317, MSC319, MSC322, MSC323, MSC324, MSC325, MSC326, MSC328, MSC346,MSC347, MSC348, MSC350, MSC356, MSC359, MSC392, MSC402, MSC403, MSC404,MSC405, MSC410, MSC411, MSC436, MSC437, MSC438, MSC439, MSC458, MSC459,MSC460, MSC461, MSC466, MSC467, MSC469, MSC480, MSC494, MSC498, MSC499,MSC500, MSC510, MSC511, MSC522, MSC526, MSC537, MSC551, MSC561, MSC562,MSC563, MSC564, MSC566, MSC567, MSC619, MSC625, MSC627, MSC640, MSC641,MSC643, MSC646, MSC650, MSC656, MSC657, MSC658, MSC659, MSC660, MSC669,MSC670, MSC671, MSC672, MSC673, MSC674, MSC675, MSC676, MSC677, MSC678,MSC679, MSC680, MSC681, MSC682, MSC683, MSC684, MSC687, MSC688, MSC689,MSC690, MSC691, MSC692, MSC693, MSC694, MSC700, MSC701, MSC702, MSC722,MSC723 and MSC724.
 78. A method for producing a non-pathogenic bacterialhost cell comprising the construct of claim 65, comprising transferringthe construct to a non-pathogenic bacterial host cell.
 79. A method forproducing a bacterial cell that is capable of producingnon-fructosylated chondroitin, wherein the cell comprises a kfoA gene, akfoC gene, and a kfoF gene, the method comprising inactivating a gene inthe cell selected from the group consisting of: kfoD, orf3(kfoI), kfoE,orf1(kfoH), and combinations thereof.
 80. A recombinant bacterial cellcomprising a kfoA gene, a kfoC gene, and a kfoF gene, wherein the celldoes not comprise a functional gene of one or more of kfoD, orf3(kfoI),kfoE, or orf1(kfoH), and wherein the cell is capable of producingchondroitin.
 81. The recombinant bacterial cell of claim 80, wherein thekfoA gene, kfoC gene, kfoF gene, or combinations thereof are expressedfrom a promoter selected from the group consisting of Pm, Plac, Ptp,Ptac, λpL, PT7, PphoA, ParaC, PxapA, Pcad, and PrecA.
 82. A geneticallymodified microorganism comprising a kfoA gene, a kfoC gene, and a kfoFgene, wherein the microorganism has been genetically modified to deleteor inactivate a gene of one or more of kfoD, orf3(kfoI), kfoE, ororf1(kfoH), and wherein the microorganism is capable of producingchondroitin.
 83. A method for producing a chondroitin, comprisingculturing the recombinant bacterial cell of claim 80 under fermentationconditions sufficient for production of the chondroitin.
 84. A methodfor producing a chondroitin, comprising culturing the geneticallymodified microorganism of claim 82 under fermentation conditionssufficient for production of the chondroitin.
 85. A method for producinga chondroitin, comprising culturing a non-pathogenic bacterial host cellcomprising the construct of claim 65 under fermentation conditionssufficient for production of the chondroitin.
 86. The method of claim85, wherein the genes of the construct are integrated into a chromosomeof the bacterial host cell.
 87. The method of claim 86, wherein two ormore copies of a gene of the construct are integrated the chromosome ofthe bacterial host cell.
 88. The method of any one of claims 83-85,wherein the chondroitin is non-fructosylated.
 89. A method for producinga chondroitin sulfate, comprising: (a) producing a chondroitin by themethod of any one of claims 83-85, and (b) sulfating the chondroitin.90. The method of claim 89, wherein the sulfating comprises mixingsulfurtrioxide-triethylamine complex or chlorosulfonic acid with thechondroitin in formamide.
 91. The chondroitin produced by the method ofany one of claims 83-85.
 92. The chondroitin sulfate produced by themethod of claim
 89. 93. A composition comprising the chondroitin ofclaim
 91. 94. A composition comprising the chondroitin sulfate of claim92.